Transglutaminase variants and applications of use thereof

ABSTRACT

Variants of the transglutaminase enzyme of  Streptomyces mobaraensis  are provided. The disclosed variants exhibit at least about 2-fold increased enzymatic activity versus the wild-type enzyme. Methods and compositions are provided for biocidal applications of use and for covalently binding small organic molecules to a protein or material of interest. Methods are provided for increasing the self-life of products, such as personal care, household and industrial products, by incorporating an effective amount of the disclosed variant enzymes into the product. The transglutaminase variants may also be used to covalently bind functional ingredients, such as UV-blocking molecules, dyes, or pigments to proteins. The transglutaminase enzymes and functional ingredients may be incorporated into a cosmetic formulation for modifying skin, hair, or nail proteins or skin-derived proteins, such as collagen, keratin, and/or elastin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.63/024,398, filed on May 13, 2020, and 63/074,288, filed on Sep. 3,2020, both of which are incorporated by reference herein in theirentireties.

FIELD OF THE INVENTION

The present invention relates to engineered variant microbialtransglutaminase polypeptides comprising two or more amino acidsubstitutions, and the nucleic acids encoding them. The variants may beused for conjugating proteins, peptides, or small molecules withincreased specific activity compared to wild-type Streptomycesmobaraensis transglutaminase, such as in topical applications of use.The variants may also be used as active biocidal enzymes and informulations thereof for use as agents for broad spectrum microbialcontrol.

BACKGROUND

Transglutaminases (Tgase, EC 2.3.2.13) are enzymes capable of catalyzingan acyl transfer reaction in which a γ-carboxy-amide group of a peptidebound glutamine residue is the acyl donor. Primary amino groups in avariety of compounds may function as acyl acceptors with the subsequentformation of monosubstituted γ-amides of peptide bound glutamine. Whenthe ε-amino group of a lysine residue in a peptide chain serves as theacyl acceptor, the Tgases form intramolecular or intermolecularγ-glutamyl-ε-lysyl crosslinks. The catalytic reaction proceeds viaglutamine deamination and formation of a protein-glutamyl-thioester atthe active site of the enzyme. Nucleophilic attack by a lysyl ε-aminogroup of a second protein at the carbonyl moiety of the thioesterintermediate generates isopeptide-crosslinked proteins that are largelyresistant to proteolysis by common peptidases (Mariniello, et al. (2007)J Agr Food Chem. 55:4717-4721). Bonds formed by a Tgase exhibit highresistance to proteolytic degradation (proteolysis). Tgases frommicrobial origin are calcium-independent, which represents a majoradvantage for their practical use.

Tgase has found many applications in biotechnology and in the foodprocessing industry, where it has earned the moniker “meat glue.” Thepeptide crosslinking activity has shown useful for a variety ofindustrial purposes ranging from food processing, biotechnology,pharmaceuticals, medical devices, personal and household goods, andleather and textile treatment. The most commonly used Tgase is microbialtransglutaminase from Streptomyces mobaraensis, having the amino acidsequence depicted in SEQ ID NO:1 and referred to hereinafter as Tgase.

Lysyl oxidases (LOX) (also known as protein-lysine 6-oxidase) arecopper-dependent enzymes that catalyze formation of aldehydes fromlysine residues in collagen and elastin precursors. These aldehydes arehighly reactive, and undergo spontaneous chemical reactions with otherlysyl oxidase-derived aldehyde residues, or with unmodified lysineresidues. This results in cross-linking collagen and elastin. LOXproteins have been identified in animals, other eukaryotes, and inbacteria and archaea (reviewed in Grau-Bove, et al. (2015) ScientificReports 5: Article number: 10568).

Semi-permanent personal care products are increasingly popular amongconsumers who want to put less effort into their daily routines.Furthermore, waterproof and sweat-proof cosmetics have been long soughtafter by consumers and product formulators to achieve long-lastingeffects of applied makeup and dyes. The use of hydrophobic silicones hasbeen extensively used to achieve longer lasting effects in this context.A disadvantage of cosmetics is the inability to sustain an initial orfreshly applied look after application. The applied product in liquidand powder makeup sits on top of the skin and therefore has the tendencyto transfer easily from the skin, lips, and/or hair onto objects withwhich it is brought into contact, such as glassware, cups, fabrics, orother skin. Long-lasting lip sticks, glosses, and stain utilizebromoacids to stain the skin, which can leave the skin dry andirritated. Moreover, the mobility of the applied product often allowsthe product to migrate and/or concentrate easily into the fine lines,wrinkles, folds, and/or pores of the skin and/or lips, resulting in anundesirable non-uniform appearance.

Conventional cosmetic foundations are typically sold in a liquid,semi-liquid or cream, or powder form. Powder-type formulations may beperceived as having a shorter useful life than the liquid-typeformulations (i.e., do not provide the desired cosmetic benefit for aslong) and/or are more susceptible to undesirable transfer from the skinto another surface. This is due to the fact that powder-type foundationproducts are essentially individual solid particles lying on the skinsurface with little to prevent them from being rubbed or wiped away.Throughout the course of the day, the product is exposed to sebum(produced by the skin), moisture (sweat, tears, humidity, rain, washing)and oils (skin care products, other cosmetic products). As such,conventional liquid and semi-liquid products are not suited for stayingon the skin for longer than one day. One drawback shared by conventionalconcealers and foundations is that they may be unintentionallytransferred to other surfaces (e.g., clothes, furniture, hair, and otherareas of the body). Such transfers may result in clothing, furniture orother surfaces being soiled with makeup, and/or it may reduce theeffective life of the makeup. A variety of mechanisms may contribute tothe undesired transfer of makeup from the skin. For example, sebum orother waxy/oily substances found on the skin can impede the ability of afoundation or concealer to adhere to the skin. In some instances,rubbing against a surface may abrade the concealer or foundation fromthe skin. In order to overcome the drawback of undesirable transferand/or to increase the effective life of a product, “long-wear” or“transfer-free” products are known. However, even conventional long-wearor transfer-free products may not provide a suitable level of coverageand/or beauty enhancement for more than 24 hours or provide suitableresistance to the abrasion encountered by skin during typical dailyactivities.

Moreover, cosmetics and other personal care products are typicallyapplied and removed during the same day. This leaves many time-points inconsumers' lives when they are not able to enjoy the benefits of theproduct.

Accordingly, it would be desirable to provide a semi-permanent cosmeticproduct that can withstand the environmental insults typically faced bya product in use for more than twenty-four hours. It would further bedesirable to provide a semi-permanent cosmetic formulation that exhibitsgood abrasion resistance. It would also be desirable to provide asemi-permanent cosmetic formulation that can withstand exposure to waterand soap during showering and facial cleansing.

A new category of cosmetic and personal care products is desired: asemi-permanent technology that provides the same effect as when theproduct is initially applied, for multiple hours or days. Such a productdoes not require daily application or the frequent touch-ups ofconventional technologies.

There is also considerable interest in binding functional or activeingredients to skin for long-lasting effects. One such application is toextend UV protection by binding of sunscreen ingredients to skinproteins or exogenous proteins for long-lasting protection and reducedskin penetration of the ingredient.

There is also considerable interest in preventing topical active orfunctional ingredients from penetrating the skin by anchoring theseingredients to a high molecular weight, cosmetically acceptable peptideor protein. For example, binding chemical sunscreen molecules to aprotein or peptide prior to or at the point of topical application willprevent these ingredients' penetration and absorption into thebloodstream (FIG. 1 ). A recent FDA monograph ruling indicated twelvechemical sunscreens demonstrated higher than acceptable levels in thebloodstream after a single application(https://www.fda.gov/news-events/fda-voices/shedding-more-light-sunscreen-absorption).Anchoring these sunscreen ingredients to a high molecular weight proteinor peptide will prevent their absorption through the skin and uptakeinto the bloodstream, enhancing the safety of chemical sunscreens forconsumers and meeting regulatory requirements.

There is also considerable interest in adding color to or dyinganimal-based products which are comprised of the same proteins as thosefound in human skin (e.g., collagen, keratin, elastin) or similarnaturally derived structural proteins (e.g., silk). For example, theability to permanently bond color to leather, silk, wool, or fabricblends thereof, without using traditional dying techniques which havethe potential to damage these natural fibers by causing undesirableshrinkage, crimping, or elongation, would be desirable. In addition, theprocess of dying these fabrics is a large source of environmentalpollutants and is driving the textile and fashion industries to identifysuitable alternative methods. It is similarly important formanufacturers to be able to dye these fabrics to design clothes andother textiles which are fashionably and visually appealing.

There is also considerable interest in the ability to enhance the colorof food products with natural dyes which do not transfer or bleed in thefinal prepared product, especially as it pertains to meat and processedmeat products. Edible food casings made of a skin fiber composition ofanimal origin (collagen) are increasingly used for the production offrankfurters and Vienna sausages, and their manufacture is described,for example, in German Pat. No. 972,854. Such collagen sausage casingsrange from practically colorless to pale yellow, which is not visuallyappealing or appetizing. In sausages where the casings are intended tobe consumed, and especially in the case of various scalding sausagessuch as bockwurst, an increasing consumer preference towards a stronglycolored sausage has become evident. Thus, in some countries such as USA,France, and the UK, certain sausages such as hot dogs are marketed dyedred.

Such a method of dying has several disadvantages. First, uniform dyingcan be obtained only with great difficulty. The casing shows regionswhich are dyed more weakly or not at all, depending on how closetogether the sausages were arranged and how adequately they were wetted.Furthermore, the dyestuff tends to both bleed out and also to migrateinto the sausage material. An improved method for dying sausage casingswould be desirable.

BRIEF SUMMARY OF THE INVENTION

Transglutaminase (Tgase) enzymes are provided herein. The enzymes arevariants of Streptomyces mobaraensis Tgase (SEQ ID NO:1). Some of thevariants demonstrate improvement in transamidation activity that atleast about 1.2-fold or at least about 2-fold, e.g., about 1.2-fold toabout 10-fold greater than the wild-type Streptomyces mobaraensis enzyme(at least about or greater than any of about 20%, 50%, 100%, 150%, 200%,250%, 300%, 350%, 400%, 450%, 500%, or 1000% improvement in enzymaticactivity).

It is desirable to have high specific activity of transglutaminase toallow for lower quantities of enzyme for cross-linking glutamine-donorsubstrates with amine substrates in the transamidation reaction, toallow for lower cost of product development. Additionally, it isbeneficial to identify mutational variants of transglutaminase thatexhibit higher initial rates to a deliver shorter reaction times. Suchvariants may promote rapid cross-linking for applications of use such asmicrobial control. One example is preservation, where rapid crosslinkingof proteins, e.g., cellular surface proteins, leads to superiormicrobial control, such as, but not limited to, faster or more effectivemicrobial kill rate. In other embodiments, the transglutaminase variantsmay be use in applications such as bonding of dye molecules or pigmentsto collagen, keratin, elastin, and/or other structural or accessoryskin, hair, or nail proteins or peptides, which may be found in aproduct formulation or on the surface of skin, hair, or nails, such asbonding agents for semi-permanent or permanent application of functionalingredients, color, dye, or pigment in long-lasting topically appliedproducts.

In one aspect, variants of the Streptomyces mobaraensis Tgase enzyme areprovided. In some embodiments, the variant comprises or consists of: (i)S199G and S299V (SEQ ID NO:2); (ii) H289V and S299A (SEQ ID NO:3); (iii)N292M, H289T, and S299V (SEQ ID NO:4); (iv) N282E, H289V, and S299K (SEQID NO:5); (v) S284D, H289L, and S299K (SEQ ID NO:6); (vi) N282E, H289I,and S299K (SEQ ID NO:7); (vii) N282K, G283A, and S299V (SEQ ID NO:8);(viii) N282Q, S284P, H289E, and S299V (SEQ ID NO:9); (ix) N282K, G283A,S284P, and S299V (SEQ ID NO:10); (x) N282R, G283A, S284E, H289Q, andS299V (SEQ ID NO:11); (xi) S199A and S299A (SEQ ID NO:12); (xii) S199Aand S299E (SEQ ID NO:13); (xiii) S199A and S299K (SEQ ID NO:14); (xiv)S199A and S299V (SEQ ID NO:15); (xv) S199G and S299A (SEQ ID NO:16);(xvi) S199G and S299K (SEQ ID NO:17); (xvii) S2P, S199A, and S299A (SEQID NO:18); (xviii) S2P, S199A, and S299E (SEQ ID NO:19); (xix) S2P,S199A, and S299K (SEQ ID NO:20); (xx) S2P, S199A, and S299V (SEQ IDNO:21); (xxi) S2P, S199G, and S299A (SEQ ID NO:22); (xxii) S2P, S199G,and S299E (SEQ ID NO:23); (xxiii) S2P, S199G, and S299K (SEQ ID NO:24);(xxiv) S2P, S199G, and S299V (SEQ ID NO:25); (xxv) N292D, G283A, S284A,and S299V (SEQ ID NO:26); or (xxvi) S199G and S299E (SEQ ID NO:27),relative to the wild-type enzyme sequence (SEQ ID NO:1), or has thedisclosed mutations and one or more conservative substitution within theremainder of the amino acid sequence, e.g., having the disclosedmutations and at least about 90%, at least about 95%, or at least about98% sequence identity to the disclosed amino acid sequence, or is acircular permutant of a variant having the disclosed mutations.

Variants of the Tgase sequence depicted in SEQ ID NO:1 are provided thatcomprise or consist of substitutions of amino acid residue A or G atposition 199 and amino acid residue A, E, K, or V at position 299, orhas the disclosed mutations and one or more conservative substitutionwithin the remainder of the amino acid sequence, e.g., having thedisclosed mutations and at least about 90%, at least about 95%, or atleast about 98% sequence identity to the disclosed amino acid sequence,or is a circular permutant of a variant having the disclosed mutations.

Variants of the Tgase sequence depicted in SEQ ID NO:1 are provided thatcomprise or consist of substitutions of amino acid residue C, D, E, F,H, I, K, L, M, N, P, Q, R, T, V, W, or Y at position 2, A or G atposition 199 and amino acid residue A, E, K, or V at position 299, orhas the disclosed mutations and one or more conservative substitutionwithin the remainder of the amino acid sequence, e.g., having thedisclosed mutations and at least about 90%, at least about 95%, or atleast about 98% sequence identity to the disclosed amino acid sequence,or is a circular permutant of a variants having the disclosed mutations.

Variants of the Tgase sequence depicted in SEQ ID NO:1 are provided thatcomprise or consist of substitutions of amino acid residue E or M atposition 282, amino acid residue I, T, or V at position 289, and aminoacid residue K or V at position 299, or has the disclosed mutations andone or more conservative substitution within the remainder of the aminoacid sequence, e.g., having the disclosed mutations and at least about90%, at least about 95%, or at least about 98% sequence identity to thedisclosed amino acid sequence, or is a circular permutant of a varianthaving the disclosed mutations.

In some embodiments, a variant as described herein further comprises aC-terminal polyhistidine sequence. In some embodiments, a variant asdescribed herein further comprises an N-terminal methionine residue. Insome embodiments, the transglutaminase is a circular permutant of any ofthe amino acid sequences described herein, optionally further comprisingan N-terminal methionine residue. In some embodiments, thetransglutaminase enzyme further includes a pro-sequence. In someembodiments, a variant as disclosed herein, or a variant havingdisclosed mutations and at least about 90%, at least about 95%, or atleast about 98% sequence identity to the disclosed amino acid sequence,or a variant that is a circular permutant of a variant having disclosedmutations, has at least about 2-fold greater transglutaminase enzymeactivity than the wild type enzyme having the amino acid sequencedepicted in SEQ ID NO:1.

In another aspect, methods are provided for increasing the shelf lifeand/or eliminating odor of a product. The methods include incorporatinga transglutaminase variant as described herein into the product in anamount effective to prevent or decrease growth of one or more microbe incomparison to an identical product that does not include thecomposition.

In another aspect, products are provided that include a transglutaminasevariant as described herein in an effective amount to increase the shelflife of the product, in comparison to an identical product that does notinclude the enzyme. For example, the product may be a personal care,household, industrial, food, pharmaceutical, cosmetic, healthcare,marine, paint, coating, energy, plastic, packaging, or agriculturalproduct. In some embodiments, the product bar soap, liquid soap, handsanitizer, preoperative skin disinfectant, cleansing wipes, disinfectingwipes, body wash, acne treatment products, antifungal diaper rash cream,antifungal skin cream, shampoo, conditioner, cosmetics deodorant,antimicrobial creams, body lotion, hand cream, topical cream, aftershavelotion, skin toner, mouth wash, toothpaste, or sunscreen lotion. Inother embodiments, the product is a wound care product selected fromwound healing ointments, creams, and lotions, wound coverings, burnwound cream, bandages, tape, or steri-strips.

In another aspect, an enzyme composition is provided that includes: (i)a transglutaminase variant enzyme as described herein, atransglutaminase enzyme comprising or consisting of the amino acidsequence depicted in SEQ ID NO:1, a transglutaminase enzyme comprisingor consisting of a; and (ii) a substrate for the transglutaminaseenzyme, such as a sunscreen molecule, functional ingredient, a pigment,or a dye molecule. In some embodiments, the sunscreen molecule,functional ingredient, pigment or dye molecule is conjugated to amolecule that includes a free amino group (FIG. 2 ). For example, themolecule that includes a free amino group may be derived fromderivatization with a linker such as lysine, cadaverine, putrescine,hydrazine, adipic acid dihydrazide, sebacic dihydrazide, orhexamethylenediamine (FIG. 4 ).

In some embodiments, the molecule that includes a free amino group isderived from an aliphatic amine of formula R(CH₂)_(n)(NH₂), wherein n isan integer between 1 and 30, or n is an integer between 5 and 10, and Ris a functional ingredient. Previous attempts at this chemicalfunctionality have focused on preparing carbamates at the phenolicoxygen, formula presented in FIG. 4A. (See, e.g., U.S. Pat. No.5,490,980). However, these compounds have not been shown to besubstrates for transglutaminase. The present disclosure has successfullydemonstrated linkers via Schiff Base reactions at the carbonyl ofoxybenzone or avobenzone with diamines separated by (CH2)_(n) (Formulain FIGS. 4B-4C) where n is an integer between 1 and 30, or between 5 and10. The present disclosure has demonstrated that these amine containingsunscreen molecules are substrates for transglutaminase.

In some embodiments, the sunscreen molecule, pigment, or dye molecule isconjugated to an amino acid, peptide, or protein with a free glutamineside chain (FIG. 3 ). Cosmetic compositions that include the enzymecomposition are also provided.

In another aspect, methods are provided for bonding color to a materialor protein of interest. The methods include contacting the material orprotein of interest with a transglutaminase variant enzyme as describedherein and a pigment or dye molecule, wherein the transglutaminasevariant enzyme is present in an amount effective to covalently bind thepigment or dye molecule to the material or protein of interest (FIG. 7and FIG. 8 ). In one embodiment, the protein of interest is a proteinthat is present in skin. For example, the protein that is present inskin may be collagen, keratin, and/or elastin.

In another aspect, products are provided that include a transglutaminasevariant enzyme as described herein in an effective amount to add a colormolecule onto a protein or a protein-, peptide-, or aminoacid-containing material of interest when contacted with the product. Insome embodiments, the product may be a personal care, cosmetic, leather,food, or agricultural product. Methods for modifying the color of aprotein or material of interest are also provided, which includecontacting the protein or material of interest with the product.

In another aspect, compositions are provided that include atransglutaminase variant enzyme as described herein in combination withone or more antimicrobial enzyme, peptide, or protein, wherein thecomposition possesses a preservative, biocidal, antimicrobial, orvirucidal activity. In some embodiments, the antimicrobial enzyme,peptide, or protein is lysozyme, chitinase, lipase, lysin, lysostaphin,glucanase, DNase, RNase, lactoferrin, glucose oxidase, peroxidase,lactoperoxidase, lactonase, acylase, dispersin B, a-amylase, cellulase,nisin, bacteriocin, siderophore, polymyxin, or defensin.

In another aspect, a bacteriophage is provided, which includes a nucleicacid sequence that encodes a transglutaminase variant enzyme asdescribed herein. In one embodiment, a composition that includes thebacteriophage provides antimicrobial activity. The composition mayfurther include a pharmaceutically acceptable excipient.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of thedisclosed methods and compositions and together with the description,serve to explain the principles of the disclosed methods andcompositions.

FIG. 1 schematically shows covalent addition of sunscreen conjugated toan aliphatic diamine (CH2)_(n)(NH₂)₂, where the sunscreen is oxybenzoneor avobenzone, to an example protein, collagen, catalyzed bytransglutaminase or lysyl oxidase to yield sunscreen-linker-collagen.The enzyme catalyzes cross-linking between an amine group on thesunscreen molecule and either a Gln sidechain (transglutaminase) or Lyssidechain (lysyl oxidase) on collagen. The enzyme can be encapsulated orunencapsulated.

FIG. 2 schematically shows covalent addition of sunscreen-linker-NH₂ toan example protein, collagen, catalyzed by transglutaminase to yieldsunscreen-linker-collagen. The enzyme catalyzes cross-linking between anamine group on the linker molecule and a Gln sidechain on collagen. Theenzyme can be encapsulated or unencapsulated.

FIG. 3 schematically shows covalent addition of sunscreen-linker-Gln toan example protein, collagen, catalyzed by transglutaminase to yieldsunscreen-linker-collagen. The enzyme catalyzes cross-linking betweenthe Gln group on the linker molecule and an available amine on a Lysresidue on collagen. The enzyme can be encapsulated or unencapsulated.

FIGS. 4A-4C illustrate formulas of sunscreen and linker molecules whichmay be combined to produce sunscreen-linker adducts. FIG. 4A shows amethod for functionalizing oxybenzone via carbamate formation at thephenolic alcohol but not demonstrated to have reactivity withtransglutaminase. FIG. 4B shows imine formation via the carbonyl ofoxybenzone produces a transglutaminase compatible amine donor. FIG. 4Cshows imine formation via the carbonyl of avobenzone produces a mixtureof two transglutaminase compatible amine donors.

FIG. 5 illustrates the reaction of 1,6-hexane diamine (HMDA) withoxybenzone followed by covalent addition of the oxybenzone-linker adductto an example protein, Cbz-Gln-Gly, catalyzed by transglutaminase toyield sunscreen-linker-protein. The enzyme catalyzes cross-linkingbetween a Gln sidechain on dipeptide and an available amine group on thesunscreen-linker. The enzyme can be encapsulated or unencapsulated.

FIG. 6 shows characterization of the reaction product of Tgase catalyzedcoupling of oxybenzone-linker to Cbz-Gln-Gly. This figure compares theamount of sunscreen-linker-protein product between wild-type Tgase,Tgase variant SEQ ID NO:28, and a negative control in the absence ofTgase. Tgase SEQ ID NO:28 contains 11-fold more product than wild-typeTgase. No sunscreen-linker-protein product is observed in the absence ofTgase.

FIG. 7 schematically shows covalent addition of Dye-NH₂(TAMRA-cadaverine) to Collagen catalyzed by transglutaminase to yieldDye-Collagen. The enzyme catalyzes cross-linking between an amine groupon the dye molecule and a Gln sidechain on collagen. The enzyme can beencapsulated or unencapsulated.

FIG. 8 schematically shows covalent addition of Dye-Gln to Collagencatalyzed by transglutaminase to yield Dye-Collagen. The enzymecatalyzes cross-linking between a Gln sidechain on the dye (ZQG-TAMRA)and a Lys, N-terminus, or other amine group on collagen. The enzyme canbe encapsulated or unencapsulated.

FIG. 9 shows an enzyme dose response of TAMRA-cadaverine (1.7 g/L) insolution covalently bound to a collagen plate catalyzed by Tgase variantSEQ ID NO:28 (0 to 0.33% w/v). Dye (TAMRA-cadaverine) was bound to thecollagen plate by increasing amounts of Tgase variant SEQ ID NO:28 (leftto right). As a negative control in the last column, Tgase was replacedby bovine serum albumin (BSA, 0.33% w/v). Bottom row shows thecollagen-bound dye after removing residual, unbound dye through awashing process with phosphate buffered saline (PBS).

FIG. 10 shows covalent addition of the short dye labeled peptideCbz-Gln-Gly-TAMRA (2.5 g/L) in solution to a collagen plate catalyzed byTgase variant SEQ ID NO:28. Column 1 illustrates the Dye(Cbz-Gln-Gly-TAMRA) in buffer added to the collagen plate in thepresence or absence of Tgase. Column 2 illustrates removal of the excessreaction mixture after incubation. Column 3 represents a washing stepwith PBS to remove unbound Dye. Column 4 represents Dye bound tocollagen after removal of excess unbound Dye. Dye was bound to thecollagen in the presence of Tgase, and minimal or no binding wasobserved in the absence of Tgase.

DETAILED DESCRIPTION

Tgase enzymes are provided herein. The enzymes are variants of Tgase(SEQ ID NO:1), and demonstrate improvements in transamidation activitythat is at least about 1.2-fold, at least about 2-fold, at least about10-fold, or at least about 11-fold greater than the wild-typeStreptomyces mobaraensis enzyme.

Also provided herein are applications of use for lysyl oxidase (LOX)enzymes, which may be utilized to form reactive aldehydes, for example,on cosmetically relevant proteins, such as collagen, to react withfunctional ingredients containing a nucleophile, such as a free R—NH₂.

The cross-linking enzymes disclosed herein may be employed as novelbiocidal agents for microbial control, with applications in healthcareproducts, personal care or cosmetic formulations, packaging (e.g., food,cosmetic, and pharmaceuticals), textile and leather production, paintsand coatings, and marine applications including water treatment andpurification. In some embodiments, Tgase enzymes disclosed herein may beemployed for permanently modifying proteins of interest, such as, butnot limited to, keratin and collagen, with dyes or proteins. In someembodiments, the Tgase enzymes may be used as preservatives.

Tgase enzymes that are mutant forms of the Streptomyces mobaraensisTgase are disclosed herein. Specifically, the enzymes described hereinare proteins obtained by mutating at least two amino acids in thepolypeptide sequence of the wild-type Tgase, or circular permutantsthereof, and observing transglutaminase transamidation activity betweena glutamine amino acid residue and an amine (or hydroxylamine) acceptor.

Methods for recombinant expression of proteins with mutationalsubstitutions have been described previously, for example, MolecularCloning, A Laboratory Manual 4th ed., Cold Spring Harbor Press (1989),Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997)and the like. Point mutant enzymes can be generated using site-directedmutagenesis or any other methods known in the art. Such methods caninclude, but are not limited to, using kits and commercially availablereagents such as the Kunkel method, KLD method, or Gapped duplex method,and examples of the kit, for example, QuickChange™ Site-DirectedMutagenesis Kit (Stratagene), GeneArt™ Site-Directed Mutagenesis System(Invitrogen), Q5®, Site-Directed Mutagenesis System (New EnglandBiolabs), TaKaRa Site-Directed Mutagenesis System (Prime STAR®Mutagenesis Basal kit, or Muta-Direct™ Site Directed Mutagenesis Kit(iNtRON), and the like.

Compositions and methods are provided herein for covalent bonding offunctional ingredients such as UV-blocking molecules and/or colorproducing molecules (such as dye and pigment molecules) to proteins andpeptides, in applications of use such as cosmetic and/or sunscreenproducts, for sunscreen and/or color application and binding to skin andskin-derived proteins and peptides, such as collagen, keratin, and/orelastin, and for color application and binding to food products such asedible food casings.

I. Definitions

Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively.

Numeric ranges provided herein are inclusive of the numbers defining therange.

“A,” “an,” and “the” include plural references unless the contextclearly dictates otherwise.

The term “about” is used herein to mean plus or minus ten percent (10%)of a value. For example, “about 100” refers to any number between 90 and110.

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified unless clearly indicated to the contrary. Thus,as a non-limiting example, a reference to “A and/or B,” when used inconjunction with open-ended language such as “comprising” can refer, inone embodiment, to A without B (optionally including elements other thanB); in another embodiment, to B without A (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

The term “amino acid” refers to a molecule containing both an aminegroup and a carboxyl group that are bound to a carbon, which isdesignated the alpha-carbon. Suitable amino 30 acids include, withoutlimitation, both the D- and L-isomers of the naturally occurring aminoacids, as well as non-naturally occurring amino acids prepared byorganic synthesis or other metabolic routes. In some embodiments, asingle “amino acid” might have multiple sidechain moieties, as availableper an extended aliphatic or aromatic backbone scaffold. Unless thecontext specifically indicates otherwise, the term amino acid, as usedherein, is intended to include amino acid analogs.

As used herein, “antimicrobial” refers to a substance that is intendedto kill or inhibit the growth of bacteria, fungi, and viruses, forexample, according to the Environmental Protection Agency (EPA).

The term “base pair” or “bp” as used herein refers to a partnership(i.e., hydrogen bonded pairing) of adenine (A) with thymine (T), or ofcytosine (C) with guanine (G) in a double stranded DNA molecule. In someembodiments, a base pair may include A paired with Uracil (U), forexample, in a DNA/RNA duplex.

A “bead” refers to a solid particle, comprising or consisting of apolymer as described herein.

As used herein, “biocide” refers to a substance that killsmicroorganisms, for example, according to the Environmental ProtectionAgency (EPA).

“Biodegradable” refers to a substance that is capable of decompositionby microbes (e.g., bacteria) or other living organisms.”

“Cbz” is an abbreviation for benzyloxycarbonyl.

The term “catalyst” refers to a chemical actor, such as a molecule ormacromolecular structure, which accelerates the speed at which achemical reaction occurs where a reactant or reactants is converted intoa product or products, while the catalyst is not turned into a productitself, or otherwise changed or consumed at the completion of thechemical reaction. After a catalyst participates in one chemicalreaction, because it is unchanged, it may participate in furtherchemical reactions, acting on additional reactants to create additionalproducts. To accelerate a chemical reaction a catalyst decreases theactivation energy barrier across the reaction path allowing it to occurat a colder temperature, or faster at a given temperature. In this way,a more rapid approach of the system to chemical equilibrium may beachieved. Catalysts subsume enzymes, which are protein catalysts.

A “circular permutant” refers to a protein that has a changed order ofamino acids in its amino acid sequence in comparison with a referencesequence. The result is a protein structure with different connectivity,but overall similar three-dimensional (3D) shape in comparison to thereference protein. For example, an N-terminal fragment may be moved tothe C-terminal end of the protein amino acid sequence.

A “coloring agent” or “color producing molecule” refers to a molecule orcompound that imparts a color to mammalian (e.g., human) skin, hair, ornails. Coloring agents may include dyes and/or pigments. Nonlimitingexamples of coloring agents include phenols, naphthols, and hydroxy azoderivatives. As discussed herein, a coloring agent may be modified toinclude an amino group (e.g., an alkylamino, alkylhydrazine,alkylhydrazide, or alkoxyamine moiety), attached either directly to thecoloring agent or indirectly via a linker that is attached to thecoloring agent.

As used herein, the term “composition” refers to a combination of two ormore substances, for example, a combination that includes one or morecross-linking enzyme as described herein and one or more chromophores,such as UV-blocking molecules, dyes, pigments, or other color-producingmolecules.

A “conservative modification” or “conservative substitution” means, inrespect of a polypeptide, the replacement of an amino acid therein withanother amino acid having a similar side chain. Families of amino acidshaving similar side chains are known in the art. Such families includeamino acids with basic side chains (lysine, arginine, histidine), acidicside chains (aspartic acid, glutamic acid), uncharged polar side chains(asparagine, glutamine, serine, threonine, tyrosine, cysteine,tryptophan), nonpolar side chains (glycine, alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine), branched side chains(threonine, valine, isoleucine), small side chains (glycine, alanine,serine), chain orientation changing side chains (glycine, proline) andaromatic side chains (tyrosine, phenylalanine, tryptophan).

The term “derived from” encompasses the terms “originated from,”“obtained from,” “obtainable from,” “isolated from,” “purified from,”and “created from,” and generally indicates that one specified materialfinds its origin in another specified material or has features that canbe described with reference to another specified material.

The term “duplex” herein refers to a region of complementarity thatexists between two polynucleotide sequences. The term “duplex region”refers to the region of sequence complementarity that exists between twooligonucleotides or two portions of a single oligonucleotide.

A “dye” refers to a colored substance, e.g., a natural or syntheticsubstance used to add a color to or change the color of something, whichis typically a water-soluble organic molecule.

“Effective amount” as used herein refers to an amount (e.g., minimuminhibitory concentration (MIC)) of a preservative composition asdisclosed herein that is sufficient to prevent or inhibit microbialgrowth. The preservative compositions described herein may be activeagainst Gram positive bacteria, Gram negative bacteria, yeasts, and/ormolds.

“Emollients” are externally applied agents that soften or soothe skin,and are generally known in the art and listed in compendia, such as the“Handbook of Pharmaceutical Excipients”, 4^(th)_Ed., PharmaceuticalPress, 2003.

“Emulsifiers” are surface active substances which promote the suspensionof one liquid in another and promote the formation of a stable mixture,or emulsion, of hydrophobic and hydrophilic substances, such as oil andwater.

“Encapsulate” or “encapsulation” as used herein refers to the entrapmentor enclosure of an enzyme in a matrix. The matrix can be polymer aloneor polymer with a cross-linking agent to covalently bind the enzyme tothe polymer or to a porous polymeric network structure of the matrix orto a semi-permeable membrane coating containing the enzyme.

As used herein, the term “expression” refers to the process by which apolypeptide is produced based on the nucleic acid sequence of a gene.The process includes both transcription and translation.

“Functional ingredient” refers to an ingredient which performs orfulfills a specific function within a product to deliver a benefit tothe consumer. For example, sunscreen molecules block UV radiation andpigment or dye molecules modify skin, hair, or nail color.

A “gene” refers to a DNA segment that is involved in producing apolypeptide and includes regions preceding and following the codingregions as well as intervening sequences (introns) between individualcoding segments (exons).

“Household products” are products, other than personal care products,that would be used by individual consumers.

“Hybridization” and “annealing” refer to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson Crick base pairing, Hoogstein binding, or inany other sequence specific manner. The complex may include two nucleicacid strands forming a duplex structure, three or more strands forming amulti-stranded complex, a single self-hybridizing strand, or anycombination of these. A hybridization reaction may constitute a step ina more extensive process, such as the initiation of polymerase chainreaction (PCR), ligation reaction, sequencing reaction, or cleavagereaction, e.g., enzymatic cleavage of a polynucleotide by a ribozyme. Afirst nucleic acid sequence that can be stabilized via hydrogen bondingwith the bases of the nucleotide residues of a second sequence is saidto be “hybridizable” to the second sequence. In such a case, the secondsequence can also be said to be hybridizable to the first sequence. Theterm “hybridized” refers to a polynucleotide in a complex that isstabilized via hydrogen bonding between the bases of the nucleotideresidues.

“Industrial products” refers to products that are used in industry.

The terms “isolated,” “purified,” “separated,” and “recovered” as usedherein refer to a material (e.g., a protein, nucleic acid, or cell) thatis removed from at least one component with which it is naturallyassociated, for example, at a concentration of at least 90% by weight,or at 15 least 95% by weight, or at least 98% by weight of the sample inwhich it is contained. For example, these terms may refer to a materialwhich is substantially or essentially free from components whichnormally accompany it as found in its native state, such as, forexample, an intact biological system. An isolated nucleic acid moleculeincludes a nucleic acid molecule contained in cells that ordinarilyexpress the nucleic acid molecule, but the nucleic acid molecule ispresent extrachromosomally or at a chromosomal location that isdifferent from its natural chromosomal location.

A “mature” polypeptide, protein or enzyme refers to the activated formof a zymogen or proprotein following cleavage of its pro-sequence or inthe absence of the pro-sequence. In some embodiments, the mature enzymemay be produced as a separate polypeptide from the pro-sequence in orderto eliminate a post-translational processing (activation) step.

A “microbead” refers to a bead that is less than one millimeter in itslargest dimension.

The terms “microorganism” and “microbe” can include bacteria, protozoa,fungi, algae, amoebas, viruses, and molds life forms.

The term “mutation” herein refers to a change introduced into a parentalsequence, including, but not limited to, substitutions, insertions, anddeletions (including truncations), thereby producing a “mutant.” Theconsequences of a mutation include, but are not limited to, the creationof a new character, property, function, phenotype or trait not found inthe protein encoded by the parental sequence.

The term “nucleotide” herein refers to a monomeric unit of DNA or RNAconsisting of a sugar moiety (pentose), a phosphate, and a nitrogenousheterocyclic base. The base is linked to the sugar moiety via theglycosidic carbon (1′ carbon of the pentose) and that combination ofbase and sugar is a nucleoside. When the nucleoside contains a phosphategroup bonded to the 3′ 30 or 5′ position of the pentose it is referredto as a nucleotide. A sequence of polymeric operatively linkednucleotides is typically referred to herein as a “base sequence,”“nucleotide sequence,” “polynucleotide sequence,” “oligonucleotidesequence”, or nucleic acid or polynucleotide “strand,” and isrepresented herein by a formula whose left to right orientation is inthe conventional direction of 5′-terminus to 3′-terminus, referring tothe terminal 5′ phosphate group and the terminal 3′ hydroxyl group atthe “5′” and “3′” ends of the polymeric sequence, respectively.

“Optional” or “optionally” means that the subsequently described event,circumstance, or material may or may not occur or be present, and thatthe description includes instances where the event, circumstance, ormaterial occurs or is present and instances where it does not occur oris not present.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein, “pathogen” refers to microorganisms (e.g., bacteria,viruses, or parasites) that can cause disease in humans, animals, and/orplants.

“Peptide” refers to a compound consisting of two or more amino acidslinked in a chain, the carboxyl group of each acid being joined to theamino group of the next by a bond of the type R—OC—NH—R′, for example,about 2 to about 50 amino acids.

As used herein, “permanent” refers to the bonded application of either asunscreen molecule or a color molecule, such as a dye or pigmentmolecule, on a surface or a protein molecule through a chemical covalentbond rather than a physical deposition or adsorption.

A “pigment” refers to a material that provides color, e.g., a materialthat changes the color of reflected or transmitted light as the resultof wavelength-selective absorption, which is typically a water insolubleinorganic substance, such as, but not limited to, a mineral or a metalsalt.

The term “polymerase” herein refers to an enzyme that catalyzes thepolymerization of nucleotides (i.e., the polymerase activity). The termpolymerase encompasses DNA polymerases, RNA polymerases, and reversetranscriptases. A “DNA polymerase” catalyzes the polymerization ofdeoxyribonucleotides. An “RNA polymerase” catalyzes the polymerizationof ribonucleotides. A “reverse transcriptase” catalyzes thepolymerization of deoxyribonucleotides that are complementary to an RNAtemplate.

The terms “polynucleotide,” “nucleic acid,” and “oligonucleotide” areused interchangeably. They refer to a polymeric form of nucleotides ofany length, either deoxyribonucleotides or ribonucleotides, or analogsthereof. Polynucleotides may have any three-dimensional structure, andmay perform any function, known or unknown, may be single- ormulti-stranded (e.g., single-stranded, double-stranded, triple-helical,etc.), and may contain deoxyribonucleotides, ribonucleotides, and/oranalogs or modified forms of deoxyribonucleotides or ribonucleotides,including modified nucleotides or bases or their analogs. Because thegenetic code is degenerate, more than one codon may be used to encode aparticular amino acid, and the present invention encompassespolynucleotides which encode a particular amino acid sequence. Any typeof modified nucleotide or nucleotide analog may be used, so long as thepolynucleotide retains the desired functionality under conditions ofuse, including modifications that increase nuclease resistance (e.g.,deoxy, 2′-O-Me,phosphorothioates, etc.). Labels may also be incorporatedfor purposes of detection or capture, for example, radioactive ornonradioactive labels or anchors, e.g., biotin. The term polynucleotidealso includes peptide nucleic acids (PNA). Polynucleotides may benaturally occurring or non-naturally occurring. Polynucleotides maycontain RNA, DNA, or both, and/or modified forms and/or analogs thereof.A sequence of nucleotides may be interrupted by non-nucleotidecomponents. One or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S (“thioate”), P(S)S (“dithioate”), (O)NR2 (“amidate”), P(O)R,P(O)OR′, CO or CH2 (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl,cycloalkenyl or araldyl. The following are nonlimiting examples ofpolynucleotides: coding or non-coding regions of a gene or genefragment, intergenic DNA, loci (locus) defined from linkage analysis,exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, shortinterfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA),small nucleolar RNA, ribozymes, cDNA, recombinant polynucleotides,branched polynucleotides, plasmids, vectors, isolated DNA of anysequence, isolated RNA of any sequence, nucleic acid probes, adapters,and primers. A polynucleotide may include modified nucleotides, such asmethylated nucleotides and nucleotide analogs. If present, modificationsto the nucleotide structure may be imparted before or after assembly ofthe polymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component,tag, reactive moiety, or binding partner. Polynucleotide sequences, whenprovided, are listed in the 5′ to 3′ direction, unless stated otherwise.

As used herein, “polypeptide” refers to a composition comprised of aminoacids and recognized as a protein by those of skill in the art. Theconventional one-letter or three-letter code for amino acid residues isused herein. The terms “polypeptide” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Also,included within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art.

As used herein, “preservative” is an agent added to a product asdescribed herein to prevent (for some period of time) the growth ofmicroorganisms, or the occurrence of undesirable chemical reactions(such as oxidation or odor generation), that spoil or deteriorate,including deterioration of one or more utility, of the product.

As used herein, the term “product” is intended to refer to a preparationor composition that has a specific utility, such as a consumer packagedgood. Examples include, but are not limited to, personal care products,household products, cosmetics, over the counter therapeutics,pharmaceutical preparations, paints, coatings, adhesives, andformulations for purchase by a consumer. In certain embodiments, aproduct includes a composition for bonding a sunscreen molecule(chromophore), dye, or pigment or modifying a protein with a sunscreenor color-producing molecule, such as a cosmetic or topically appliedproduct.

A “promoter” refers to a regulatory sequence that is involved ininitiating transcription of a gene by RNA polymerase. A promoter may bean inducible promoter or a constitutive 5 promoter. An “induciblepromoter” is a promoter that is active under environmental ordevelopmental regulatory conditions.

A “pro-sequence” refers to a polypeptide sequence within an expressedprotein, e.g., a zymogen or proprotein, such as transglutaminase, whichis typically cleaved from the protein to produce an active protein, suchas an enzyme. In some embodiments, a pro-sequence may be essential forcorrect folding of the protein. In some embodiments, cleavage of thepro-sequence results in transition of an inactive enzyme to activeenzyme. In some embodiments, the pro-sequence may be cleaved at multiplepositions within the proprotein sequence, which may result in improvedenzyme activity or stability relative to the native mature enzymesequence.

The term “recombinant,” refers to genetic material (i.e., nucleic acids,the polypeptides they encode, and vectors and cells comprising suchpolynucleotides) that has been modified to alter its sequence orexpression characteristics, such as by mutating the coding sequence toproduce an altered polypeptide, fusing the coding sequence to that ofanother gene, placing a gene under the control of a different promoter,expressing a gene in a heterologous organism, expressing a gene at adecreased or elevated levels, expressing a gene conditionally orconstitutively in manner different from its natural expression profile,and the like. Generally recombinant nucleic acids, polypeptides, andcells based thereon, have been manipulated such that they are notidentical to related nucleic acids, polypeptides, and cells found innature. A recombinant cell may also be referred to as “engineered.”

A “reversibly soluble polymer” refers to a polymer which can phasetransition from a soluble to insoluble material in solution in responseto controllable stimuli in the environment, such as, but not limited to,pH, temperature, or ionic strength. This transition process can berepeatably cycled between phases.

“Shelf life” refers to the length of time for which an item (e.g., aproduct as described herein) remains usable, fit for consumption, orsaleable.

The phrases “substantially similar” and “substantially identical” in thecontext of at least two nucleic acids typically means that apolynucleotide includes a sequence that has at least about 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99.5% sequenceidentity, in comparison with a reference (e.g., wild-type)polynucleotide or polypeptide. Sequence identity may be determined usingknown programs such as BLAST, ALIGN, and CLUSTAL using standardparameters. (See, e.g., Altshul et al. (1990) J. Mol. Biol. 215:403-410;Henikoff et al. (1989) Proc. Natl. Acad. Sci. 89:10915; Karin et al.(1993) Proc. Natl. Acad. Sci. 90:5873; and Higgins et al. (1988) Gene73:237). Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information. Also,databases may be searched using FASTA (Pearson et al. (1988) Proc. Natl.Acad. Sci. 85:2444-2448.) In some embodiments, substantially identicalnucleic acid molecules hybridize to each other under stringentconditions (e.g., within a range of medium to high stringency). Nucleicacid “synthesis” herein refers to any in vitro method for making a newstrand of polynucleotide or elongating an existing polynucleotide (i.e.,DNA or RNA) in a template dependent manner. Synthesis, according to theinvention, can include amplification, which increases the number ofcopies of a polynucleotide template sequence with the use of apolymerase. Polynucleotide synthesis (e.g., amplification) results inthe incorporation of nucleotides into a polynucleotide (e.g., extensionfrom a primer), thereby forming a new polynucleotide moleculecomplementary to the polynucleotide template. The formed polynucleotidemolecule and its template can be used as templates to synthesizeadditional polynucleotide molecules. “DNA synthesis,” as used herein,includes, but is not limited to, polymerase chain reaction (PCR), andmay include the use of labeled nucleotides, e.g., for probes andoligonucleotide primers, or for polynucleotide sequencing. “Undertranscriptional control” is a term well understood in the art thatindicates that transcription of a polynucleotide sequence depends on itsbeing operably linked to an element which contributes to the initiationof, or promotes transcription.

“Surfactants” are surface-active agents that lower surface tension andthereby increase the emulsifying, foaming, dispersing, spreading andwetting properties of a product.

The term “sunscreen” refers to an organic compound or mixture of organiccompounds that can protect and block human skin from ultraviolet A (UVA)and/or ultraviolet B (UVB) radiation. Nonlimiting examples ofUV-absorbing sunscreen compounds include benzoates, oxybenzones, andcinnamic acid. Exemplary organic sunscreens include, but are not limitedto, para-aminobenzoic acid, trolamine salicylate, cinoxate,dioxybenzone, ensulizole, homosalate, meradimate, octinoxate,octisalate, octocrylene, padimate O, sulisobenzone, oxybenzone, andavobenzone. Sunscreens may be approved by the Food and DrugAdministration (FDA) or other international regulatory bodies for thepurpose of blocking UVA and/or UVB radiation. Sunscreens may also bemolecules which provide UVA and/or UVB protection, but which are not yetapproved by the FDA or other international regulatory bodies for thepurpose of blocking UVA and/or UVB radiation. Sunscreens may also bemolecules with analogous structures to those approved by the FDA orother international regulatory bodies and which also provide UVA and/orUVB protection (sunscreen analogs). Sunscreens may also be modified bylinkers and/or other small molecules and still provide UVA and/or UVBprotection, either with the linker attached or after hydrolysis(sunscreen-linker adducts). A “sunscreen” herein may refer to asunscreen molecule, a sunscreen analog, and/or a sunscreen-linkeradduct. As discussed herein, a sunscreen molecule may be modified toinclude an amino group (e.g., an alkylamino, alkylhydrazine,alkylhydrazide, or alkoxyamine moiety), attached either directly to thesunscreen molecule or indirectly via a linker (e.g., as part of alinker) that is attached to the sunscreen molecule.

“TAMRA” is an abbreviation for carboxytetramethylrhodamine.

Related (and derivative) proteins encompass “variant” proteins. Variantproteins differ from another (i.e., parental) protein and/or from oneanother by a small number of amino acid residues. A variant may includeone or more amino acid mutations (e.g., amino acid deletion, insertionor substitution) as compared to the parental protein from which it isderived. Alternatively or additionally, variants may have a specifieddegree of sequence identity with a reference protein or nucleic acid,e.g., as determined using a sequence alignment tool, such as BLAST,ALIGN, and CLUSTAL (see, infra). For example, variant proteins ornucleic acid may have at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or even 99.5% amino acid sequence identity with areference sequence.

A “zymogen” or “proenzyme” refers to an inactive precursor of an enzyme,which may be converted into an active enzyme by catalytic action, suchas via proteolytic cleavage of a pro-sequence.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. The methods andtechniques of the present disclosure are generally performed accordingto conventional methods well-known in the art. Generally, nomenclaturesused in connection with, and techniques of biochemistry, enzymology,molecular and cellular biology, microbiology, genetics and protein andnucleic acid chemistry and hybridization described herein are thosewell-known and commonly used in the art. The methods and techniques ofthe present disclosure are generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification unless otherwise indicated.

II. Transglutaminase Variants

Tgase enzymes are provided herein that are variants of theCa²⁺-independent microbial transglutaminase (Tgase) Streptomycesmobaraensis. Tgase variants with at least about 1.2-fold (20%) or 2-fold(100%) improvement in enzyme activity, versus the wild-type enzyme fromStreptomyces mobaraensis (SEQ ID NO:1), are disclosed herein.

The amino acid sequence of wild-type S. mobaraensis Tgase is provided inSEQ ID NO:1. Tgase variants as described herein may be obtained bymutating at least two amino acids in the polypeptide sequence of thewild-type Tgase, and observing transglutaminase transamidation activitybetween a glutamine amino acid residue and an amine (or hydroxylamine)acceptor. Methods for recombinant expression of proteins with mutationalsubstitutions have been described previously and are well known in theart, for example, Molecular Cloning, A Laboratory Manual 4th ed., ColdSpring Harbor Press (1989), Current Protocols in Molecular Biology, JohnWiley & Sons (1987-1997) and the like. Combinations of point mutationscan be generated using a number of methods including error-prone PCR,gene shuffling, molecular breeding, and the like.

The amino acid sequences of Tgase variants, relative to the wild-typeamino acid sequence set forth in SEQ ID NO:1, are disclosed in Table 3,along with enzymatic activity improvement, relative to the wild-typeenzyme (SEQ ID NO:1).

One Tgase variant of this invention, designated M2, has a doublemutation (S199G and S299V), relative to the sequence of the wild-type S.mobaraensis Tgase (SEQ ID NO: 1). The amino acid sequence of variant M2is shown in SEQ ID NO: 2.

One Tgase variant of this invention, designated M3, has a doublemutation (H289V and S299A), relative to the sequence of the wild-type S.mobaraensis Tgase. The amino acid sequence of variant M3 is shown in SEQID NO: 3.

One Tgase variant of this invention, designated M4, has a triplemutation (N282M, H289T and S299V), relative to the sequence of thewild-type S. mobaraensis Tgase. The amino acid sequence of variant M4 isshown in SEQ ID NO: 4.

One Tgase variant of this invention, designated M5, has a triplemutation (N282E, H289V, and S299K), relative to the sequence of thewild-type S. mobaraensis Tgase. The amino acid sequence of variant M5 isshown in SEQ ID NO: 5.

One Tgase variant of this invention, designated M6, has a triplemutation (S284D, H289L, and S299K), relative to the sequence of thewild-type S. mobaraensis Tgase. The amino acid sequence of variant M6 isshown in SEQ ID NO: 6.

One Tgase variant of this invention, designated M7, has a triplemutation (N282E, H289L, and S299K), relative to the sequence of thewild-type S. mobaraensis Tgase. The amino acid sequence of variant M7 isshown in SEQ ID NO: 7.

One Tgase variant of this invention, designated M8, has a quadruplemutation (N282K, G283A, and S299V), relative to the sequence of thewild-type S. mobaraensis Tgase. The amino acid sequence of variant M8 isshown in SEQ ID NO: 8.

One Tgase variant of this invention, designated M9, has a quadruplemutation (N282Q, S284P, H289E, and S299V), relative to the sequence ofthe wild-type S. mobaraensis Tgase. The amino acid sequence of variantM9 is shown in SEQ ID NO: 9.

One Tgase variant of this invention, designated M10, has a quadruplemutation (N282K, G283A, S284P, and S299V), relative to the sequence ofthe wild-type S. mobaraensis Tgase. The amino acid sequence of variantM10 is shown in SEQ ID NO: 10.

One Tgase variant of this invention, designated M11, has a five-mutation(N282R, G283A, S284E, H289Q, and S299V), relative to the sequence of thewild-type S. mobaraensis Tgase. The amino acid sequence of variant M11is shown in SEQ ID NO: 11.

One Tgase variant of this invention, designated M12, has a doublemutation (S199A and S299A), relative to the sequence of the wild-type S.mobaraensis Tgase. The amino acid sequence of variant M12 is shown inSEQ ID NO: 12.

One Tgase variant of this invention, designated M13, has a doublemutation (S199A and S299E), relative to the sequence of the wild-type S.mobaraensis Tgase. The amino acid sequence of variant M13 is shown inSEQ ID NO: 13.

One Tgase variant of this invention, designated M14, has a doublemutation (S199A and S299K), relative to the sequence of the wild-type S.mobaraensis Tgase. The amino acid sequence of variant M14 is shown inSEQ ID NO: 14.

One Tgase variant of this invention, designated M15, has a doublemutation (S199A and S299V), relative to the sequence of the wild-type S.mobaraensis Tgase. The amino acid sequence of variant M15 is shown inSEQ ID NO: 15.

One Tgase variant of this invention, designated M16, has a doublemutation (S199G and S299A), relative to the sequence of the wild-type S.mobaraensis Tgase. The amino acid sequence of variant M16 is shown inSEQ ID NO: 16.

One Tgase variant of this invention, designated M17, has a doublemutation (S199G and S299K), relative to the sequence of the wild-type S.mobaraensis Tgase. The amino acid sequence of variant M17 is shown inSEQ ID NO: 17.

One Tgase variant of this invention, designated M18, has a triplemutation (S2P, S199A, and S299A), relative to the sequence of thewild-type S. mobaraensis Tgase. The amino acid sequence of variant M18is shown in SEQ ID NO: 18.

One Tgase variant of this invention, designated M19, has a triplemutation (S2P, S199A, and S299E), relative to the sequence of thewild-type S. mobaraensis Tgase. The amino acid sequence of variant M19is shown in SEQ ID NO: 19.

One Tgase variant of this invention, designated M20, has a triplemutation (S2P, S199A, and S299K), relative to the sequence of thewild-type S. mobaraensis Tgase. The amino acid sequence of variant M20is shown in SEQ ID NO: 20.

One Tgase variant of this invention, designated M21, has a triplemutation (S2P, S199A, and S299V), relative to the sequence of thewild-type S. mobaraensis Tgase. The amino acid sequence of variant M21is shown in SEQ ID NO: 21.

One Tgase variant of this invention, designated M22, has a triplemutation (S2P, S199G, and S299A), relative to the sequence of thewild-type S. mobaraensis Tgase. The amino acid sequence of variant M22is shown in SEQ ID NO: 22.

One Tgase variant of this invention, designated M23, has a triplemutation (S2P, S199G, and S299E), relative to the sequence of thewild-type S. mobaraensis Tgase. The amino acid sequence of variant M23is shown in SEQ ID NO: 23.

One Tgase variant of this invention, designated M24, has a triplemutation (S2P, S199G, and S299K), relative to the sequence of thewild-type S. mobaraensis Tgase. The amino acid sequence of variant M24is shown in SEQ ID NO: 24.

One Tgase variant of this invention, designated M25, has a triplemutation (S2P, S199G, and S299V), relative to the sequence of thewild-type S. mobaraensis Tgase. The amino acid sequence of variant M25is shown in SEQ ID NO: 25.

One Tgase variant of this invention, designated M26, has a quadruplemutation (N282D, G283A, S284A, and S299V), relative to the sequence ofthe wild-type S. mobaraensis Tgase. The amino acid sequence of variantM26 is shown in SEQ ID NO: 26.

One Tgase variant of this invention, designated M27, has a doublemutation (S199G and S299E), relative to the sequence of the wild-type S.mobaraensis Tgase. The amino acid sequence of variant M27 is shown inSEQ ID NO: 27.

Variants M2-M27 can have conservative substitutions thereto, providedtheir respective distinctive substitutions: (i) S199G/S299V, (ii)H289V/S299A, (iii) N292M/H289T/S299V, (iv) N282E/H289V/S299K, (v)S284D/H289L/S299K, (vi) N282E/H289I/S299K, (vii) N282K/G283A/S299V,(viii) N282Q/S284P/H289E/S299V, (ix) N282K/G283A/S284P/S299V, (x)N282R/G283A/S284E/H289Q/S299V, (xi) S199A/S299A, (xii) S199A/S299E,(xiii) S199A/S299K, (xiv) S199A/S299V, (xv) S199G/S299A, (xvi)S199G/S299K, (xvii) S2P/S199A/S299A, (xviii) S2P/S199A/S299E, (xix)S2P/S199A/S299K, (xx) S2P/S199A/S299V, (xxi) S2P/S199G/S299A, (xxii)S2P/S199G/S299E, (xxiii) S2P/S199G/S299K, (xxiv) S2P/S199G/S299V, (xxv)N292D/G283A/S284A/S299V, or (xxvi) S199G/S299E, respectively, arepreserved. Such conservatively modified versions of variants M2-M27 areincluded in the scope of this invention. Plural conservativesubstitutions/modifications may be present. Conservatively modifiedversions of variants M2-M27 may be at least about 90%, 95%, or 98%identical to their respective unmodified sequences and may have at leastabout 2-fold greater transglutaminase enzymatic activity than thewild-type transglutaminase (SEQ ID NO:1).

Tgase variants disclosed herein (e.g., M2-M27) may further comprise apolyhistidine peptide extension at their C-terminus, as exemplified withamino acid residues 334-339 of SEQ ID NO:29. The polyhistidine peptideis a useful tag for purification purposes and does not affect enzymaticactivity. Typically, the polyhistidine peptide is 6-8 residues long.

Tgase variants disclosed herein (e.g., M2-M27) may further comprise amethionine residue at their N-terminus. The mature wild-typeStreptomyces mobaraensis Tgase enzyme lacks the N-terminal methionineresidue encoded by the gene sequence that encodes the enzyme. In someembodiments, the Tgase variant is expressed as a variant of the matureStreptomyces mobaraensis Tgase without an N-terminal methionine residue.In other embodiments, the Tgase is expressed as the mature Tgase with anadditional N-terminal methionine residue, which may be provided by anexpression vector from which the Tgase is expressed.

Tgase variants herein may further include a pro-sequence. In someembodiments, the variant is expressed with a pro-sequence, either aspart of the variant polypeptide sequence (e.g., an additional amino acidsequence as an extension of an amino acid sequence described in Table 3)or as a separate polypeptide. In some embodiments, the mature variantpolypeptide is expressed in the presence of a polypeptide Tgasepro-sequence. In one embodiment, a DNA sequence that encodes thepro-sequence and the DNA sequence that encodes the mature Tgase variantare expressed as discrete polypeptide sequences from the same DNAtemplate. In another embodiment, the DNA sequence that encodes themature polypeptide is expressed from a first DNA template, and the DNAsequence that encodes the pro-sequence is expressed from a separatesecond DNA template. In another embodiment, the pro-sequence issynthesized chemically and added to an expression system prior to,during, or after expression of the mature polypeptide. In one example,the Tgase variant may be expressed in a cell free expression system, asdisclosed in PCT Application No. US20/49226, which is incorporated byreference herein in its entirety.

In some embodiments, the Tgase variant is expressed, e.g., expressedrecombinantly, with a homologous pro-sequence, i.e., the nativepro-sequence for the Tgase enzyme, i.e., the pro-sequence for thewild-type Tgase enzyme from the same organism. In other embodiments, theTgase variant is expressed, e.g., expressed recombinantly, with aheterologous pro-sequence, i.e., a pro-sequence for the same enzyme butfrom a different organism or a pro-sequence for a different enzyme fromthe same or different organism.

In some embodiments, a Tgase variant may be a circular permutant of aTgase variant described herein (e.g., a variant described in Table 3).In some embodiments, the Tgase variant may be a circular permutant of aTgase variant as described in Table 3, optionally further including anN-terminal methionine residue. The circular permutants may provide novelsubstrate specificities, product profiles, and reaction kinetics versusthe parent enzyme, i.e., the wild-type enzyme or a disclosed variant,e.g., as depicted in Table 3. A circular permutant retains the samebasic folding of the parent enzyme, but has the N-terminus in adifferent position, with the original N- and C-termini connected,optionally by a linking sequence. In a Tgase wild-type or variantcircular permutant, the N-terminal residue of the wild-type or variantenzyme is positioned at a site in the protein other than the naturalN-terminus.

III. Antimicrobial Compositions

Disclosed are compositions, e.g., biocidal, preservative, antimicrobial,anti-bacterial, and anti-viral (virucidal) compositions that include oneor more Tgase variant enzyme as described herein, such as any of thevariants disclosed in Table 3, optionally with an N-terminal methionineresidue, including circular permutants thereof, and optionally with apro-sequence as described herein. Such a composition may be included inor with (e.g., within or associated with) products to be preserved,e.g., for microbial control. The Tgase variant enzyme may catalyze areaction of amino acid residues on a protein, thereby effecting, forexample, protein cross-linking or binding a molecule of interest to aprotein. In some embodiments, the compositions include one or more Tgasevariant enzyme, e.g., comprising or consisting of one or more Tgasevariant as disclosed herein, in an amount effective to inhibit microbial(e.g., bacterial) growth, e.g., inhibition of 80% to 100%, or any of atleast about 80%, 85%, 90%, 95%, 98%, or 99% of microbial growth, in aproduct to be preserved.

Preservatives are antimicrobial ingredients added to productformulations to maintain the microbiological safety of the products byinhibiting the growth of and reducing the amount of microbialcontaminants. US Pharmacopeia has published protocols for acceptablemicrobial survival for preservatives in cosmetics and personal careproducts. These tests include USP 51 (Antimicrobial Effectiveness Test)and USP 61 (Microbial Limits Test) (https://www.fda.gov/files/about%20fda/published/Pharmaceutical-Microbiology-Manual.pdf).

The effectiveness of the preservative system disclosed herein isdetermined based on the MIC (minimum inhibitory concentration) against avariety of microbes, including, but not limited to, Gram positivebacteria, Gram negative bacteria, yeast and/or mold (e.g. E. coli DH10B,E. coli ATCC 8739, B. subtilis BGSC 1A976, C. albicans ATCC 10231,and/or A. brasiliensis ATCC 16404). Minimum inhibitory concentrations(MICs) are defined as the lowest concentration of an antimicrobial thatwill inhibit the growth of a microorganism. Microbial growth may bedetermined, for example, by spectrophotometric methods (the opticaldensity at 600 nm) or with a cell viability assay (BacTiter Glo,Promega).

In some embodiments, the compositions include one or more additionalbiocidal enzymes, such as a cross-linking enzyme, oxidase, nuclease,hydrolase, protease, and/or lytic enzyme. In some embodiments, thecomposition further includes one or more biocidal chemical, such as, butnot limited to, chitosan, polylysine, and/or quaternary ammoniumcompounds. Exemplary, but nonlimiting examples of biocidal enzymes,compositions, and formulations, and methods of use thereof, aredisclosed in PCT/US20/21211, which is incorporated by reference hereinin its entirety.

Without wishing to be bound by theory, the use of a biocidal enzymeenhances the antimicrobial properties of a biocidal chemical byproviding an additional mechanism of antimicrobial action. Chitosan, forexample, ruptures the cell membrane and leads to spillage of the cellcontents. The cross-linking Tgase enzyme can cross-link proteins vitalfor cell function both on the surface of the cell and within the cell.This combination of both materials together reduce the quantity of thematerials needed and provide additional stability to the enzyme allowingfor greater activity over time (less chitosan and less enzyme) andreduce the undesirable effects that may accompany the use of biocidalchitosan.

A. Biocidal Proteins and Peptides

In some embodiments, the compositions include one or more antimicrobialpeptides. Examples of antimicrobial peptides include, but are notlimited to, nisin and pediocin.

In some embodiments, the compositions include one or more antimicrobialproteins. Examples of antimicrobial proteins include, but are notlimited to, casein.

Nonlimiting examples of known biocidal enzymes and antimicrobialpeptides, which may be included in combination with a Tgase variantenzyme as disclosed herein, are shown in Table 1. In some embodiments, aTgase variant enzyme as described herein may be utilized in a biocidal,preservative, anti-bacterial, or anti-viral (virucidal) composition incombination with one or more of the antimicrobial enzymes, peptides, orproteins described in Table 1.

TABLE 1 Enzymes, Peptides and Proteins with Known AntimicrobialProperties Mechanism Enzyme Description Citation Lytic Lysozyme Producedby animals as part of the Ibrahim et al. (2001) innate immune system.FEBS Letters Hydrolyzes the peptidoglycan 506(1): 27-32; subunits in thebacterial cell wall. Małaczewska et al. (2019) BMC Vet. Res. 15: 318Chitinase Secreted by soil bacteria including Martínez-Zavala et alBacillus thuringiensis to combat (2020) Front. Microbiol. insects andfungi 10: 3032 Lipase Hydrolyzes extracellular lipids and Prabhawathi etal. polymers. (2014) PLoS One 9(5) Lysin Utilized by bacteriophages toHoops et al. (2008) Appl. hydrolyze the glycan component of Environ.Microbiol. bacterial cell wall 75: 5, 1388-1394 LysostaphinMetalloendopeptidase which Kokai-Kun et al. (2003) cleaves thepentaglycine bridges Antimicrob Agents found in cell wall peptidoglycan.Chermother 47(5): 1589-1597 Glucanase Secreted by soil bacteriaincluding Shafi et al. (2017) Bacillus species to degrade theBiotechnology & fungal cell wall. Has also been Biotechnologicalutilized as an algicide and for Equipment 31: 3 446-459 biofilm control.Nuclease DNase Hydrolyzes extracellular nucleic Kaplan et al. (2012)acids and viral genomic DNA. J. Antibiot. (Tokyo) 65(2): 73-77 RNaseHydrolyzes viral RNA. Wirth (1992) WO1994000016A1 Lactoferrin Sequestersessential iron ions to Niaz et al. (2019) prevent microbial growth. AlsoInternational Journal of possesses nuclease activity and Food Properties22: 1 hydrolyzes biofilm polymers. 1626-1641 Oxidoreductase GlucoseOxidase Oxidizes glucose to D-glucono-δ- Wong et al. (2008) Appl lactoneand hydrogen peroxide. Microbiol Biotechnol. 78(6): 927-938 PeroxidaseOxidizes inert substrates to form Ihalin et al. (2006) Arch. biocidalactives. Biochem. Biophys. 445, 261-268 Lactoperoxidase Oxidizes inertsubstrates to form White et al. (1983) biocidal actives. AntimicrobAgents Chemother 32(2): 267-272 Quorum Lactonase Hydrolyzes quorumsensing Schwab et al. (2019) Quenching lactones, preventing activationof Front Microbiol. 10: 611 biofilm- and pathogenesis- promotingpathways. Acylase Hydrolyzes quorum sensing Vogel et al. (2020)lactones, preventing activation of Front. Chem. 8: 54 biofilm- andpathogenesis- promoting pathways. Hydrolase Dispersin B Hydrolyzesbiofilm polymers Izano et al. (2007) J Dent Res 86(7): 618-622 α-amylaseHydrolyzes extracellular Craigen et al. (2011) polysaccharides. OpenMicrobiol J. 5: 21-31 Cellulase Hydrolyzes the cellulose Loiselle et al.(2003) component of biofilms and algal Biofouling 19(2): 77-85 cellwalls. Antimicrobial Nisin Increases permeability of the Li et al.(2018) Appl Peptides microbial cell membrane. Environ Microbiol 18(12)Bacteriocin Modes of action include inhibition Meade et al. (2020) ofcell wall synthesis and Antibiotics 9(1): 32 increasing cell membranepermeability. Siderophore Binds to and sequesters iron ions Raaska etal. (1999) J Indust Microbiol Biotechnol 22, 27-32 Polymyxin Increasespermeability of the Poirel et al. (2017) Clin microbial cell membrane.Microbiol Rev 30: 577-596 Defensin Increases permeability of the Gans(2003) Nat Rev microbial cell membrane. Immunol 3, 710-720

B. Biocidal Chemicals

In some embodiments, a Tgase variant as described herein may beformulated with one or more biocidal chemical, including, but notlimited to chitosan, polylysine, or quaternary ammonium compounds, forexample, for use as a biocidal, preservative, anti-bacterial, oranti-viral (virucidal) composition. Nonlimiting examples of biocidalchemicals are shown in Table 2.

TABLE 2 Examples of Biocidal Chemicals for Antimicrobial ApplicationsClassification Chemical Polymers Chitosan N,N,N-trimethyl chitosanε-poly-lysine Polyvinylbenzyl-dimethylbutyl ammonium chloridePolyvinylbenzyl trimethyl ammonium chloride Quaternary ammoniumpolyethyleneimine Quaternary phosphonium modified epoxidized naturalrubber Arginine-tryptophan-rich peptide Guanylated polymethacrylateAmmonium ethyl methacrylate homopolymers Metallo-terpyridinecarboxymethyl cellulose Poly(n-vinylimidazole) modified silicone rubberQuaternary Cocoamidopropyl Betaine AmmoniumMyristamidopropyl-pg-dimonium Cl Phosphate Benzalkonium ChlorideQuaternium-6 Coco Betaine Detergents Sodium Lauryl SulfateDodecylbenzenesulfonic Acid Chaotropic Polyamidopropyl biguanide AgentGuanidinium chloride Organic Acids Lactic Acid Citric Acid SalicylicAcid Sorbic Acid Acetic Acid Dehydroacetic Acid Peracetic Acid BenzoicAcid Phenols & Ethanol Alcohols Isopropanol Dichlorobenzyl AlcoholGlycerol Caprylyl Glycol Ethylhexylglycerin Benzyl Alcohol2-Phenoxyethanol Aldehydes & Glutaraldehyde Aldehyde FormaldehydeReleasers Sodium Hydroxymethylglycerate DMDM Hydantoin Base SodiumHydroxide Oxidizers Hydrogen Peroxide Parabens Methyl Paraben EthylParaben Propyl Paraben Misc Natamycin Benzisothiazolinone BronopolSorbitan Caprylate Ethyl Lauroyl Arginate MethylisothiazolinoneCetylpyridinium Chloride Chlorphenesin Zinc Omamide Sodium OmamideN-(3-aminopropyl)-N-dodecylpropane- 1,3-diamineMethylchloroisothiazolinone 2,2-dibromo-3-nitrilopropionamide1-Octadecanaminium, N,N-dimethyl-N-[3- (trimethoxysilyl)propyl]-,chloride Saponin Sodium Benzoate

1. Quaternary Ammonium Compounds

Quaternary ammonium compounds containing biopolymers, like chitosan andits more acetylated form chitin, are well known for their antimicrobialactivity (Kong, et al. (2010) Int. J. of Food Microbiol. 144: 51-63).The antimicrobial activity of chitin, chitosan and their derivativesagainst different groups of microorganisms, such as bacteria, yeast, andfungi, is known.

Quaternary ammonium compounds (non-limiting examples include, cetylpyridinium chloride, benzethonium chloride, benzalkonium chloride,polyaminopropyl biguanide), have limited use for personal care industrydue to specific incompatibilities with other cosmetic ingredients.

Lonza's Geogard series of preservative blends avoids use of parabens intheir new creations (Geogard 233S, Geogard 233S, Geogard 233S, Geogard361) however, these antimicrobial compositions are based on cationicbenzethonium chloride which gets deactivated by many anionic ingredientsthat form important part of topical personal care formulations.

2. Aldehydes & Aldehyde-Releasing Compounds

Formaldehyde is classified as Category 3 CMR (carcinogenic, mutagenicand reproductive toxicity). However, it is interesting to note that afew antimicrobials that slowly release formaldehyde are still being usedand being commercially manufactured. Due to the paucity of effective andwell-accepted antimicrobials, the industry is forced to continue withthe use of formaldehyde donors like DMDM hydantoin (CAS 6440-58-0),imidazolidinyl urea, and diazolidinyl urea (CAS 39236-46-9). Theformaldehyde released by these substances is capable of reacting withseveral cosmetic ingredients via its very reactive aldehydic carbonylfunctionality. For example, the only available and globally approvedUV-A absorber, Avobenzone, reacts with formaldehyde that is released byformaldehyde derivatives. This is a significant disadvantage forsunscreen formulations. Preservative blends, Clariant's Niapaguard PDUand Cognis's Elestab 305, ISP's Germaben II, Germaben H-E, exploitcombinations of parabens with diazolidinyl urea. ISP's Germall Plus andLonza's Glydant Plus utilize diazolidinyl urea along with iodopropynylbutyl carbamate (IPBC). McIntyre's Paragon series has DMDM hydantoin andother antimicrobials like paraben, phenoxy ethanol and IPBC. Symrise'sNeo-Dragocide and Thor's Microcare IMP exploit synergy between parabensand imidazolidinyl urea.

3. Parabens

Parabens are esters of p-hydroxy benzoic acid. Paraben compounds includein particular Methyl-paraben (CAS 99-76-3), Ethyl-paraben (CAS120-47-8), Propyl-paraben (CAS 94-13-3), Butyl-paraben (CAS 94-26-8),Isopropyl-paraben (CAS 4191-73-5), and Benzyl-paraben (CAS 94-18-8).Clariant's ‘Phenonip’ is a blend of six antimicrobials out of which thefive are parabens. The same company offers blends of only parabens as‘Nipastat’ and ‘Nipasept’, Cognis's Elestab FL 15, Elestab 48, Elestab50J, Elestab 305, Elestab 388, Elestab 3344, Elestab 4112, Elestab 4121,Elestab 4150 Lipo are all blends of antimicrobials with at least oneparaben in them. Induchem's Uniphen P23, ISP's Germaben and LiquaParseries of blends contain several parabens. Galaxy Surfactants offersGalguard NK1 and Galguard NK2 blends that are based on four and fiveparaben blends respectively with phenoxy ethanol. Five blends byMcIntyre/Rhodia from their ‘Paragon’ series have several parabens.Neolone MXP of Rohm and Haas has parabens with methyl isothiazolinone.Neo-Dragocide series of blends from Symrise has parabens. Euxyl K 300 ofSchulke and Mayr has five parabens. Thor's Microcare PM4 and MicrocarePM5 have four and five parabens respectively. Parabens are phenolderivatives; all phenolic antimicrobials have phenolic ‘hydroxyl’ groupand that is a very reactive organic functionality with very acidichydrogen with pKa of 10.

4. Halogenated Compounds

Nalco's Merguard series (four blends) relies on halogenated molecules,methyl dirbromo glutaronitrile and 2-bromo-2-nitro-1,3-diol. Severalblends of Euxyl series from Schulke and Mayr are based onchlorothiazolinones, methyl dibromo glutaronitrile,2-bromo-2-nitro-1,3-diol and diazolidinyl urea. Microcare series fromThor employs parabens, 2-bromo-2-nitro-1,3-diol, iodopropynylbutylcarbamate (IPBC), imidazolidynyl urea, and diazolidinyl urea.

The other examples of halogenated antimicrobials are chlorphenesin, andchlorhexidine. It is common knowledge that like phenolic compounds, thehalogenated organic molecules exhibit significant levels of toxiceffects. For example, IPBC has risk of thyroid hormonal disturbances dueto its iodine content. It has not been allowed in Japan and in the EU isallowed only up to 0.02% in leave-on products. Similarly, the EU permitsusage of methyl dirbromo glutaronitrile only up to 0.1% in rinse-offproducts only. Bronopol, 2-bromo-2-nitropropane-1,3-diol, is implicatedin generation of carcinogenic nitrosoamines on interacting with some ofthe nitrogen containing cosmetic ingredients. The antimicrobial efficacyof methyl chloro isothiazolinone is so powerful that it is allowed onlyin rinse-off products at 15 ppm concentration. Chloromethylisothiazolinone does have a very broad spectrum of anti-microbialactivity, but the toxicity of such powerful anti-microbials is extremelyhigh and hence cosmetic formulators do not prefer to use this kind ofpowerful antimicrobial in the cosmetics that remain on human skin for along time. It is reasonable to expect that any strong bactericide at alow concentration (ppm level) is likely to be equally lethal to anyother cells of a living organism, including human cells. This is theprecise reason why in Japan chloromethyl isothiazolinone is not allowedfor preservation if the product is going to come in contact with themucous membrane.

Halogenated compounds include 2,4-dichlorobenzyl-alcohol, Chloroxylenol(also known as 4-chloro-3,5-dimethyl-phenol, Bronopol (also known as2-bromo-2-nitropropane-1,3-diol, iodopropynyl butyl carbamate.

C. Vector Delivery

The compositions described herein may include vectors (e.g.,bacteriophage), for the delivery of genetic material encoding one ormore biocidal enzyme(s) (e.g., Tgase variant(s)) as described herein.

As used herein, “bacteriophage” and “phage” are used interchangeably torefer to a bacteriophage isolate in which members of the isolate havesubstantially the same genetic makeup, such as sharing at least aboutany of 90%, 95%, 99%, 99.9% or more sequence identity in the genome.“Bacteriophage” or “phage” refers to the parent bacteriophage as well asthe progeny or derivatives (such as genetically engineered versions)thereof. The bacteriophage can be a naturally occurring phage isolate,or an engineered phage, including vectors, or nucleic acids that encodeat least all essential genes, or the full genome of a phage to carry outthe life cycle of the phage inside a host bacterium.

IV. Compositions for Cross-Linking Functional Molecules to Protein

Disclosed are enzyme compositions that can be included in products to beused for long-lasting application of functional ingredients includingUV-blocking sunscreens, and/or coloring agents, such as pigments ordyes. The disclosed compositions include one or more cross-linkingenzyme, such as a Tgase enzyme (e.g., a Tgase variant as disclosedherein) and/or a lysyl oxidase enzyme, for the purpose of reacting aminoacid residues on skin protein or other protein-, peptide-, or aminoacid-containing materials with a molecule of interest, such as asunscreen, or color producing molecule, e.g., a pigment or dye. In someembodiments, the composition includes any of the Tgase enzymes disclosedin SEQ ID NOs:1-29, and/or a lysyl oxidase enzyme. In some embodiments,the Tgase and/or lysyl oxidase enzyme(s) acts as a catalyst to crosslinkthe active or functional ingredient (e.g., sunscreen molecule orcoloring agent) to a protein or peptide, such as a protein or peptide ofmammalian (e.g., human) skin, hair, or nails. In some embodiments, thetransglutaminase enzyme (e.g., Tgase variant enzyme) and/or lysyloxidase enzyme(s) acts as a catalyst to crosslink an amino group (e.g.,an alkylamino, alkylhydrazine, alkylhydrazide, or alkoxyamine moiety) ofthe active or functional ingredient or of a linker with an amino acid(e.g., side chain of glutamine or lysine residues) in skin, hair, ornail proteins or peptides.

Examples of skin, hair, or nail proteins include but are not limited tocollagen, keratin, elastin, and/or cornified cell envelope proteinsincluding involucrin, loricrin, small proline-rich proteins, periplakin,envoplakin, and filaggrin. Examples of peptides include but are notlimited to hydrolyzed collagen, hydrolyzed keratin, and/or hydrolyzedelastin. In some embodiments, the compositions include UV-blockingmolecules such as sunscreens. In some embodiments, the compositionsinclude coloring agents (color producing molecules), such as dye orpigment molecules. In some embodiments, the enzyme is immobilized on orencapsulated in a polymeric support. In some embodiments, the enzyme isa transglutaminase enzyme.

In some embodiments, a composition described herein includes apharmaceutically or cosmetically acceptable vehicle or carrier, e.g., toact as a diluent or dispersant for the active or functional ingredientand the Tgase and/or lysyl oxidase enzyme(s), or cross-linked active orfunctional ingredient with proteins or peptides, in the composition, forexample, to promote or facilitate distribution of the active orfunctional ingredient and the cross-linking enzyme, or cross-linkedactive or functional ingredient with proteins or peptides, when thecomposition is applied to the skin, hair, or nails of a subject. Thepharmaceutically or cosmetically acceptable vehicle or carrier maycomprise or consist of water, and may include liquid or solidemollients, solvents, humectants, thickeners, and/or powders, and insome embodiments, may form about 10% to about 99.9%, or about 50% toabout 99%, by weight of the composition. In some embodiments, thecomposition is in the form of an emulsion, which may contain an oil oroily material in an amount up to about 90%, or about 10% to about 80% byvolume of the composition. In some embodiments, the composition includesone or more emulsifier and/or one or more surfactant.

In some embodiments, the active or functional ingredient includes atleast one alkyl-amino (—RNH₂), hydrazine, hydrazide, or hydroxylaminemoiety, either directly on the active or functional ingredient, orindirectly on a linker attached (e.g., covalently bound) thereto. Forexample, the alkyl (R) group may be an aliphatic hydrocarbon chainincluding 1 to 8 carbon atoms.

In certain embodiments, a composition for application to mammalian(e.g., human) skin, hair, or nails is provided, which includes: (a) aneffective amount of at least one active or functional ingredient (suchas, for example, a sunscreen molecule or coloring agent); and (b) one ormore transglutaminase enzyme (e.g., a Tgase variant as described hereinor any of the Tgase enzymes disclosed in SEQ ID NOs:1-29, and/or a lysyloxidase enzyme) in an amount effective to catalyze the crosslinking ofthe active or functional ingredient to a protein or peptide of mammalian(e.g., human) skin, hair, or nails. In some embodiments, the compositionis formulated for topical application to skin, hair, or nails of amammalian (e.g., human) individual, and in certain embodiments maycontain: (c) a pharmaceutically or acceptable carrier in an amounteffective to deliver the Tgase and/or lysyl oxidase enzyme(s) and theactive or functional ingredient to the skin, hair, or nails of theindividual. For example, the active or functional ingredient may includeat least one alkylamino (—RNH₂), hydrazine, hydrazide, or hydroxylaminemoiety, either directly on the active or functional ingredient, orindirectly on a linker attached (e.g., covalently bound) thereto.

A. Color Molecules

The compositions described herein may contain one or more colorproducing molecule, such as a dye or pigment molecule, for applicationand binding to a surface, such as binding to one or more protein on thesurface of skin, such as collagen, keratin, and/or elastin, or bindingto an edible casing for a food product, such as a sausage casing.Nonlimiting examples of color producing molecules are described in“Summary of Color Additives for Use in the United States in Foods,Drugs, Cosmetics, and Medical Devices,” US Food and Drug Administration,https://www.fda.gov/industry/color-additive-inventories/summary-color-additives-use-united-states-foods-drugs-cosmetics-and-medical-devices.

B. Sunscreen Molecules and Linkers

The compositions described herein may contain one or more UV-blockingmolecule(s), such as a sunscreen molecule, for application and bindingto a protein or peptide within the product formulation or on the surfaceof skin, such as collagen, keratin, elastin, hydrolyzed collagen,hydrolyzed keratin, and/or hydrolyzed elastin.

Nonlimiting examples of sunscreen and/or sunscreen analog moleculesinclude but are not limited to, para-aminobenzoic acid, trolaminesalicylate, cinoxate, dioxybenzone, ensulizole, homosalate, meradimate,octinoxate, octisalate, octocrylene, padimate O, sulisobenzone,oxybenzone, avobenzone, and benzophenone hydrazone.

In some embodiments, the sunscreen is functionalized with a linkermolecule to provide a substrate handle for enzymatic binding to aprotein or peptide. A non-limiting example of this functionalization maybe accomplished through formation of a Schiff base between the sunscreenmolecule and linker. A non-limiting example of this functionalizationmay be accomplished through formation of a carbamate linkage between thesunscreen molecule and linker. The linker may include an available aminefor enzyme recognition in the form of a primary amine, hydrazine,hydrazide, or alkoxyamine moiety. The linker may also include aglutamine residue for enzyme recognition. The linker may consist of twofunctional chemical end groups linked by an aliphatic carbon chain ofvarying length for in situ formation of the sunscreen-linker molecule.Nonlimiting examples of linkers include cadaverine, putrescine,hydrazine, adipic acid dihydrazide, sebacic dihydrazide, andhexamethylenediamine.

In some embodiments, the sunscreen-linker adduct is bound to a proteinor peptide of interest and the sunscreen can be subsequently released byhydrolysis. In one embodiment, the sunscreen molecule is hydrolysable orotherwise releasable from the linker. In some embodiments, thesunscreen-linker adduct remains bound to a protein or peptide, e.g., aprotein or peptide present on skin, to provide UV-blocking protection.

C. Proteins and Peptides

The compositions described herein may contain one or more proteins orpeptides of interest for sunscreen, skin care, and/or cosmetic productsor applications of use.

Nonlimiting examples of proteins and peptides of interest for skin careproducts and cosmetics are: collagen, hydrolyzed collagen, keratin,hydrolyzed keratin, elastin, hydrolyzed elastin, silk, hydrolyzed silk,silk fibroin peptide, acetyl hexapeptide-3, acetyl hexapeptide-8, acetyltetrapeptide-5, acetyl tetrapeptide-9, acetylarginyltryptophyldiphenylglycine, copper tripeptide-1, CT-2, dipeptide-2, heptapeptide-7,hexanoyl dipeptide-3 norleucine acetate, hexapeptide-9, hexapeptide-11,manganese tripeptide-1, myristoyl hexapeptide-16, myristoylhexapeptide-16, myristoyl pentapeptide-17, nonapeptide-1, palmitoyldipeptide-5 diaminobutyroyl Hydroxythreonine, palmitoyl dipeptide-5diaminohydroxybutyrate, palmitoyl hexapeptide-12, palmitoylhexapeptide-14, palmitoyl hexapeptide-6, palmitoyl pentapeptide-4,palmitoyl tetrapeptide-7, palmitoyl tripeptide-1, palmitoyltripeptide-3, palmitoyl tripeptide-38, pentapeptide-3, pentapeptide-18,sh-oligopeptide-1, sh-oligopeptide-2, sh-polypeptide-1,sh-polypeptide-11, sh-polypeptide-9, soybean peptide, tetrapeptide PKEK,tetrapeptide-21, tripeptide-1, tripeptide-10 citrulline, and modifiedhydrolysates of hemoglobin, rice, soy, wheat protein, corn, fibronectin,reticulin, serum protein, wheat gluten.

The compositions described herein may contain one or more model peptidesof interest. One non-limiting example of a model peptide of interestincludes Cbz-Gln-Gly.

V. Polymeric Supports

In certain embodiments, compositions described herein include apolymeric support. One or more Tgase enzyme (e.g., Tgase variant enzyme)as described herein and/or lysyl oxidase enzyme is immobilized on thesupport, with or without a linker, or encapsulated within a polymericsupport, such as a reversibly soluble polymer, including, but notlimited to, chitosan, carboxymethylchitosan, or polylysine. Nonlimitingexamples of polymeric supports include: chitin, chitosan,carboxymethylchitosan, oxidized cellulose, quaternary ammoniumcellulose, alginates, pectin, and carboxycellulose. Examples ofpolymeric supports and immobilization or encapsulation of cross-linkingenzymes therein are described in PCT/US20/21211, which is incorporatedherein by reference in its entirety.

In some embodiments, the Tgase (e.g., Tgase variant enzyme as disclosedherein) and/or lysyl oxidase enzyme(s) are immobilized on particles,e.g., chitosan particles, such as beads, e.g., chitosan beads (e.g.,microbeads), or nanoparticles. For example, the beads (e.g., microbeads)may be biodegradable. In some embodiments, the enzyme may be immobilizedby encapsulation with free monomers (e.g., chitosan orcarboxymethylchitosan monomers), for example, utilizing a linker.

Chitosan is a linear aminopolysaccharide of glucosamine andN-acetylglucosamine units and is obtained by alkaline deacetylation ofchitin extracted from the exoskeleton of crustaceans such as shrimps andcrabs, as well from the cell walls of some fungi. Chitin is a linearpolymer of (1→4)-linked 2-acetamido-2-deoxy-β-D-glucopyranose (GlcNAc;A-unit), which is insoluble in aqueous solvents. It also has manystructural similarities with cellulose, such as conformation of themonomers and diequatorial glycosidic linkages. Chitosan may beconsidered as a family of linear binary copolymers of (1β4)-linkedA-units and 2-amino-2-deoxy-β-D-glucopyranose (GlcN; D-unit).Carboxymethylchitosan (e.g., of fungal origin), e.g.,N,O-carboxymethylchitosan, is >80% substituted with carboxyl groups.

Quaternary ammonium containing biopolymers, like chitosan and its moreacetylated form chitin, are well known for their antimicrobial activity(Kong, et al. (2010) Int. J of Food Microbiol. 144: 51-63). Theantimicrobial activity of chitin, chitosan and their derivatives againstdifferent groups of microorganisms, such as bacteria, yeast, and fungi,is known.

Chitin, chitosan, and other related polymers are excellent scaffolds toimmobilize enzymes (Muzzarelli (1980) Enzyme Microb. Technol.2:177-184). Tyrosinase has been immobilized on chitosan fordephenolization of industrial waste (Dinçer, et al. (2012) Int. Biol.Macromol. 50:815-820) and for optical detection of phenol compounds(Abdullah, et al. (2006) Sensors and Actuators B: Chemical 114:604-609).In these examples, the tyrosinase is either directly ligated to thechitosan support without a linker or using glutaraldehyde as a linker toimmobilize the enzyme on chitosan. Additionally, tyrosinase-chitosanbiocatalysts have been explored for the production of L-DOPA (Carvalho,et al., Appl. Biochem. Biotechnol. (2000) 84-86:791-800). Microbialtransglutaminase has been immobilized on chitosan using glutaraldehydeas a linker for the purpose of deamidation of food proteins (Nonaka, etal. (1996) Biosci, Biotechnol, and Biochem. 60:532-533), usingChitopearl 3007, a microbead form of chitosan, for the polymer support,with glutaraldehyde as a linker. Examples of polymeric supports areprovided in, for example, Nonaka, et al. (1996) Biosci, Biotechnol, andBiochem. 60:532-533 and Hayashi, T et al. (1991) J Appl Polymer Sci 42:85-92, which is incorporated by reference herein in its entirety.

A. Linkers for Enzyme Immobilization on Polymeric Support

In some embodiments, one or more Tgase enzyme (e.g., Tgase variantenzyme as disclosed herein) and/or a lysyl oxidase enzyme is immobilizedon a polymer, via a chemical linker, which covalently links the enzymeto the polymer. In some embodiments, the linker is an alkylene (e.g.methylene), a diimine (1,5-diimine), a diamine (1,5-diamine), dicarbonyl(e.g. 1,4-dicarbonyl), an amide bond, a polypeptide, an alkyl linker, orcontains a phenyl group, a fused heterocycle, or an aromatic group.

Examples of reagents which can be used to provide linkers include, butare not limited to: formaldehyde, glutaraldehyde, succinate anhydride,phenolic compounds, genipin, carbodiimide reagents, proteins or peptides(e.g., zein, gelatin, collagen).

In some embodiments the linking reagent is genepin, epichlorohydrin,formaldehyde, or glutaraldehyde.

In some embodiments, the Tgase (e.g., Tgase variant enzyme) and/or lysyloxidase enzyme(s) are covalently linked to a carrier (polymericsupport), without the use of a linker.

VI. Products

In some embodiments, products disclosed herein include personal careproducts, household products, industrial food, pharmaceutical, cosmetic,healthcare, marine, paints, coatings, adhesives, energy, plastic,packaging, or agricultural products, optionally immobilized on orencapsulated in a polymeric support, which include an effective amount,for example, about 0.0001% w/v to about 5% w/v, of one or more Tgasevariant enzyme as described herein, or a composition thereof asdescribed herein, to act as an antimicrobial agent, e.g., preservative,in the product.

In some embodiments, products disclosed herein include cosmetics andpersonal care products which include compositions described herein,compositions that include one or more Tgase enzyme (e.g., Tgase variantenzyme as described herein) and/or lysyl oxidase enzyme, optionallyimmobilized on or encapsulated in a polymeric support, and one or moreactive or functional ingredient which may include a sunscreen and/orcolor producing molecule, in an amount effective to bond a sunscreenmolecule or color to a surface, such as covalently binding to one ormore skin-derived protein or peptide either on the surface of skin orwithin the product formulation, e.g., collagen, keratin, and/or elastin.In some embodiments, the product composition includes any of the Tgaseenzymes disclosed in SEQ ID NOs:1-29, and/or a lysyl oxidase enzyme. Insome embodiments, an effective amount of the Tgase (e.g., Tgasevariant(s)) and/or lysyl oxidase is in a range of about 0.0001% to about5% w/v, such as about 0.001% to about 1% or about 0.01% to 0.1%, byweight of the composition. In some embodiments, the active or functionalingredient (e.g., sunscreen molecule or coloring agent) is present inthe composition in an amount effective to provide a benefit, such as aUV protecting benefit or a cosmetic or aesthetic benefit, e.g., to hair,skin, or nail proteins or peptides or to the hair, skin, or nails of anindividual to whom the composition is topically applied. In someembodiments, the effective amount of the active or functional ingredient(e.g., sunscreen molecule or coloring agent is in a range of about 0.1%to about 70%, such as about 1% to about 35%, by weight of thecomposition.

In some embodiments, one or more Tgase variant as described herein isincluded in a personal care product, such as, but not limited to, barsoap, liquid soap (e.g., hand soap), hand sanitizer (including rinse offand leave-on alcohol based and aqueous-based hand disinfectants),preoperative skin disinfectant, cleansing wipes, disinfecting wipes,body wash, acne treatment products, antifungal diaper rash cream,antifungal skin cream, shampoo, conditioner, cosmetics (including butnot limited to liquid or powder foundation, liquid or solid eyeliner,mascara, cream eye shadow, tinted powder, “pancake” type powder to beused dry or moistened, make up removal products, etc.), deodorant,antimicrobial creams, body lotion, hand cream, topical cream, aftershavelotion, skin toner, mouth wash, toothpaste, sunscreen lotion, and babyproducts such as, but not limited to, cleansing wipes, baby shampoo,baby soap, and diaper cream. In some embodiments, one or more Tgasevariant is included in a wound care item, such as, but not limited to,wound healing ointments, creams, and lotions, wound coverings, burnwound cream, bandages, tape, and steri-strips, and medical articles suchas medical gowns, caps, face masks, and shoe-covers, surgical drops,etc. In some embodiments, one or more Tgase variant is included in anoral care product, such as mouth rinse, toothpaste, or dental flosscoating, a veterinary or pet care product, a preservative composition,or a surface disinfectant, such as a disinfectant solution, spray orwipe.

In some embodiments, one or more Tgase variant as described herein isincorporated into a household or industrial product, for example, as apreservative substance. For example, the Tgase variant(s) may beincluded in a household cleaner, such as concentrated a liquid cleaneror spray cleaner, cleaning wipes, dish washing liquid, dish washerdetergent, spray-mop liquid, furniture polish, indoor paint, outdoorpaint, dusting spray, laundry detergent, fabric softener, rug/fabriccleaner, window and glass cleaner, toilet bowl cleaner, liquid/creamcleanser, etc. In some embodiments, one or more Tgase variant may beincluded in a food wash product, e.g., designed to clean fruits andvegetables prior to consumption, packaging, and food coatings.

Other products into which Tgase variants as described herein may beincorporated include, but are not limited to, food, pharmaceutical,cosmetic, healthcare, marine, paint, coating, energy (e.g., frackingfluid), plastic, packaging, and agricultural products. In someembodiments, the Tgase variant may be incorporated into HVAC systems,cooling ponds, water purification systems, or may be used in anindustrial application, such as, but not-limited to, pulp and paperprocessing.

Products disclosed herein include cosmetics and personal care productswhich include a Tgase enzyme (e.g., Tgase variant as described herein)and/or lysyl oxidase enzyme, or composition thereof as described herein,and one or more color producing molecule, in an amount effective to bondcolor to a surface, such as covalently binding to one or more protein ofskin, e.g., collagen, keratin, and/or elastin, or to a protein of a foodproduct, such as an edible casing for a processed food product, e.g., asausage casing. In some embodiments, the product composition includesany of the Tgase enzymes disclosed in SEQ ID NOs:1-29, and/or a lysyloxidase enzyme. In some embodiments, an effective amount of the Tgaseenzyme (e.g., Tgase variant enzyme) and/or lysyl oxidase enzyme is up toabout 1% w/v.

Products disclosed herein may include one or more Tgase variantenzyme(s), optionally immobilized on or encapsulated in a polymericsupport, and one or more functional ingredients including a sunscreenand/or color producing molecule, in an amount effective to bond asunscreen and/or color molecule to a surface. In some embodiments, theproduct covalently binds sunscreen molecules to skin-derived proteins,e.g. collagen, keratin, and/or elastin, found within the productformulation. In some embodiments, the product covalently binds sunscreenmolecules to skin proteins, e.g. collagen, keratin, and/or elastin. Insome embodiments, the product covalently binds color to skin proteins,e.g. collagen, keratin, and/or elastin. In some embodiments, the productcontains the functional ingredient with a linker which is sufficient toreact with the native enzymes on the skin's surface to crosslink toskin's proteins. In some embodiments, the product covalently binds colorto a protein of a food product, such as an edible casing for a processedfood product, e.g., a sausage casing. In some embodiments, an effectiveamount of the cross-linking enzyme (e.g., a transglutaminase enzyme) isin a range of about 0.001 to about 20% w/v, such as about 0.01% to about5% by weight of the composition

In some embodiments, a product or composition which includes Tgasevariant as described herein, further includes one or more additionalenzymes selected from acyl transferases, alpha-amylases, beta-amylases,alpha-galactosidases, arabinosidases, aryl esterases,beta-galactosidases, carrageenases, catalases, cellobiohydrolases,cellulases, chondroitinases, cutinases, endo-beta-1,4-glucanases,endo-beta-mannases, esterases, exo-mannanases, galactanases,glucoamylases, hemicellulases, hyaluronidases, keratinases, laccases,lactases, ligninases, lipases, lipoxygenases, mannanases, oxidases,pectate lyases, pectin acetyl esterases, pectinases, pentosanases,peroxidases, phenoloxidases, phosphatases, phospholipases, phytases,polygalacturonases, beta-glucanases, tannases, xylan acetyl-esterases,xylanases, xyloglucanases, xylosidases, metalloproteases, serineproteases, or combinations thereof.

In some embodiments, a Tgase variant enzyme, such as any of the variantsdisclosed in Table 3, optionally with an N-terminal methionine residue,including circular permutants thereof, and optionally with apro-sequence as described herein, or a composition thereof as describedherein, is included as an antimicrobial agent in any of the productsdisclosed herein at a concentration of any of at least about 0.0001%w/v, 0.0005% w/v, 0.001% w/v, 0.005% w/v, 0.01% w/v, 0.05% w/v, 0.1%w/v, 0.5% w/v, 1% w/v, 1.5% w/v, 2% w/v, 2.5% w/v, 3% w/v, 3.5% w/v, 4%w/v, 4.5% w/v, or 5% w/v. In some embodiments, the Tgase variant enzymeof composition thereof is included at a concentration of any of about0.0001% w/v to about 0.0005% w/v, about 0.001% w/v to about 0.005% w/v,about 0.005% w/v to about 0.01% w/v, about 0.01% w/v to about 0.05% w/v,about 0.05% w/v to about 0.1% w/v, about 0.1% w/v to about 0.5% w/v,about 0.5% w/v to about 1% w/v, about 1% w/v to about 1.5% w/v, about1.5% w/v to about 2% w/v, about 2% w/v to about 2.5% w/v, about 2.5% w/vto about 3% w/v, about 3% w/v to about 3.5% w/v, about 3.5% w/v to about4% w/v, about 4% w/v to about 4.5% w/v, about 4.5% w/v to about 5% w/v,about 0.0001% w/v to about 0.001% w/v, about 0.001% w/v to about 0.01%w/v, about 0.01% w/v to about 0.1% w/v, about 0.1% w/v to about 1% w/v,about 1% w/v to about 2.5% w/v, about 2.5% w/v to about 5% w/v, or about1% w/v to about 5% w/v.

In some embodiments, products in which a Tgase variant enzyme orcomposition thereof as described herein is included as an antimicrobialagent do not include a petrochemically derived preservative substance,such as, but not limited to, parabens, formaldehyde and formaldehydereleasers, isothiazolinones, phenoxyethanol, and/or organic acids (suchas sodium benzoate). In some embodiments, a Tgase variant enzyme asdescribed herein, alone or in combination with a biocidal chemical,e.g., chitosan, is the only antimicrobial, e.g., antibacterial orpreservative, agent in the product. In some embodiments, a Tgase variantenzyme as described herein is included as an antimicrobial agent incombination with one or more additional antimicrobial agent(s), such as,but not limited to, one or more petrochemically derived preservativesubstance(s). In some embodiments, a Tgase variant enzyme as describedherein is included as an antimicrobial agent in combination with one ormore additional antimicrobial agent(s), such as, but not limited to, oneor more petrochemically derived preservative substance(s).

In some embodiments, preservative blends are compatible with products,stable towards oxidizing or reducing agents and to normal range of pH(4.5 to 8.0) of various products.

Non-limiting examples of products in which the Tgase variants describedherein may be incorporated are described in PCT Application No.PCT/US20/21211, and in U.S. Provisional Application No. 63/075,763,which are incorporated herein by reference in their entireties.

A. Personal Care Products

A Tgase variant enzyme as described herein or composition thereof, e.g.,preservative composition, as described herein can be incorporated intoany personal care product. Personal care products into which thedisclosed Tgase variant enzymes compositions may be incorporatedinclude, but are not limited to, bar soap, liquid soap (e.g., handsoap), hand sanitizer (including rinse off and leave-on alcohol basedand aqueous-based hand disinfectants), preoperative skin disinfectant,cleansing wipes, disinfecting wipes, body wash, acne treatment products,antifungal diaper rash cream, antifungal skin cream, shampoo,conditioner, cosmetics (including but not limited to liquid or powderfoundation, liquid or solid eyeliner, mascara, cream eye shadow, tintedpowder, “pancake” type powder to be used dry or moistened, make upremoval products, etc.), deodorant, antimicrobial creams, body lotion,hand cream, topical cream, aftershave lotion, skin toner, mouth wash,toothpaste, sunscreen lotion, and baby products such as, but not limitedto, cleansing wipes, baby shampoo, baby soap, and diaper cream. Thepresent subject matter may also be applied to wound care items, such as,but not limited to, wound healing ointments, creams, and lotions, woundcoverings, burn wound cream, bandages, tape, and steri-strips, andmedical articles such as medical gowns, caps, face masks, andshoe-covers, surgical drops, etc. Additional personal care productsinclude, but are not limited to, oral products such as mouth rinse,toothpaste, dental floss coatings, veterinary and pet care products,preservative compositions, and surface disinfectants, includingsolutions, sprays or wipes.

In general, a Tgase variant enzyme as disclosed herein can beincorporated into any suitable personal care product intended for use inmodifying the appearance of skin, such as a cosmetic product (e.g.,lipstick, foundation, blush, or eye makeup). Cosmetic products intowhich the disclosed compositions may be incorporated include, but arenot limited to, liquid or powder foundation, liquid or solid eyeliner,blush, eye shadow, tinted powder, “pancake” type powder to be used dryor moistened, lip color, or makeup setting sprays, etc. The disclosedcompositions may also be incorporated into a bronzer or artificialtanning product. Additionally, the disclosed compositions may beincorporated into a sunscreen product, such as a chemical sunscreen,e.g., to bind a sunscreen chromophore (such as, but not limited to,oxybenzone avobenzone, octisalate, octocrylene, homosalate, oroctinoxate, or a derivative thereof) to skin protein.

In some embodiments, the personal care products that are protected fromthe microbial contamination by the disclosed enzymes and compositionscan be of any type of such as emulsions, gels, serums, solutions,toners, lotions, creams, spray, gel, powder, stick and cleansers.

The personal care product formulation typically includes a baseformulation to which the enzyme composition of the present disclosure isadded. The base formulation may contain numerous and differentingredients depending upon the end use application. The personal careproduct formulation, for instance, may contain solvents, surfactants,emulsifiers, consistency factors, conditioners, emollients, skin careingredients, moisturizers, thickeners, lubricants, fillers,antioxidants, other preservatives, active ingredients, in particulardermatologically active ingredients, fragrances and the like, as well asmixtures thereof. Active ingredients as mentioned herein include, forexample, anti-inflammatories, and optionally, anti-bacterials,antifungals and the like agents. In some embodiments, active ingredientssuited for topical applications are included.

In some embodiments, the personal care product does not contain anyadditional preservatives, such as a petrochemical derived preservativesubstance. In some embodiments, the personal care product includes oneor more additional preservative substance, such as a petrochemicalderived preservative, in addition to the enzyme or enzyme/polymercomposition described herein.

In some embodiments, the personal care product does not includeconventional anti-bacterial and/or antifungal “active agents” that aretypically included in personal care products. Conventionalanti-bacterials used in hand soap include: Cloflucarban, Fluorosalan,Hexachlorophene, Hexylresorcinol, Iodine complex (ammonium ether sulfateand polyoxyethylene sorbitan monolaurate), Iodine complex (phosphateester of alkylaryloxy polyethylene glycol), Nonylphenoxypoly(ethyleneoxy) ethanoliodine, Poloxamer-iodine complex, Povidone,Undecoylium chloride iodine complex, Methylbenzethonium chloride,Phenol, Phenol 16, Secondary amyltricresols, Sodium oxychlorosene,Tribromsalan, Triclocarban, Triclosan, and Triple dye. Conventionalantimicrobials used as preservatives in consumer product formulationsinclude: parabens, formaldehyde and formaldehyde releasers,isothiazolinones, phenoxyethanol, and organic acids (such as sodiumbenzoate).

In some embodiments, a Tgase variant enzyme as described herein, aloneor in combination with (e.g., blend) a biocidal chemical, including butnot limited to, chitosan, is the only antibacterial, antifungal,antimicrobial, or preservative agent in the product. In someembodiments, the Tgase variant enzyme, alone or in combination (e.g.,blend) a biocidal chemical, such as but not limited to, chitosan, iscombined with one or more additional preservative substance, such as oneor more petrochemically derived preservative substance. In someembodiments, one or more biobased preservative (i.e., Tgase variantenzyme or composition thereof as disclosed herein) is combined with oneor more synthetic preservative (e.g., petrochemical derived substance)and the preservative (e.g., antimicrobial) effect achieved between thebiobased and synthetic preservatives is additive or synergistic. In someembodiments, one or more biobased preservative (i.e., Tgase variantenzyme or composition thereof as disclosed herein) is combined with oneor more additional preservative substance, for example, a biocidalsubstance selected from polylysine, chitosan, benzoate, nisin, lysozyme,and chitosan, or any combination thereof, and the preservative (e.g.,antimicrobial) effect achieved between the biobased preservative and theadditional preservative substance(s) is additive or synergistic.

In some embodiments, the personal care product may include emollients.Emollients include, without limitation, almond oil, castor oil,ceratonia extract, cetostearoyl alcohol, cetyl alcohol, cetyl esterswax, cholesterol, cottonseed oil, cyclomethicone, ethylene glycolpalmitostearate, glycerin, glycerin monostearate, glyceryl monooleate,isopropyl myristate, isopropyl palmitate, lanolin, lecithin, lightmineral oil, medium-chain triglycerides, mineral oil and lanolinalcohols, petrolatum, petrolatum and lanolin alcohols, soybean oil,starch, stearyl alcohol, sunflower oil, xylitol and combinationsthereof. In one embodiment, the emollients are ethylhexylstearate andethylhexyl palmitate.

Common emulsifiers are: metallic soaps, certain animal and vegetableoils, and various polar compounds. Suitable emulsifiers include acacia,anionic emulsifying wax, calcium stearate, carbomers, cetostearylalcohol, cetyl alcohol, cholesterol, diethanolamine, ethylene glycolpalmitostearate, glycerin monostearate, glyceryl monooleate,hydroxpropyl cellulose, hypromellose, lanolin, hydrous, lanolinalcohols, lecithin, medium-chain triglycerides, methylcellulose, mineraloil and lanolin alcohols, monobasic sodium phosphate, monoethanolamine,nonionic emulsifying wax, oleic acid, poloxamer, poloxamers,polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives,polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates,propylene glycol alginate, self-emulsifying glyceryl monostearate,sodium citrate dehydrate, sodium lauryl sulfate, sorbitan esters,stearic acid, sunflower oil, tragacanth, triethanolamine, xanthan gumand combinations thereof. In one embodiment, the emulsifier is glycerolstearate.

Suitable non-ionic surfactants include emulsifying wax, glycerylmonooleate, polyoxyethylene alkyl ethers, polyoxyethylene castor oilderivatives, polysorbate, sorbitan esters, benzyl alcohol, benzylbenzoate, cyclodextrins, glycerin monostearate, poloxamer, povidone andcombinations thereof. In one embodiment, the non-ionic surfactant isstearyl alcohol.

Suitable antioxidants include, e.g., sulfites (e.g., sodium sulfite),tocopherol or derivates thereof, ascorbic acid or derivates thereof,citric acid, propyl gallate, chitosan glycolate, cysteine, N-acetylcysteine plus zinc sulfate, thiosulfates (e.g. sodium thiosulfate),polyphenols glutathione, dithiothreitol (DTT), superoxide dismutase,catalase and the like.

Chelators, such as ethylene diamine tetraacetic acid (EDTA), may also beincluded.

Suitable thickeners include, e.g., acrylates/steareth-20 methacrylatecopolymer, carbomer, carboxymethyl starch, cera alba, dimethicone/vinyldimethicone crosspolymer, propylene glycol alginate,hydroxyethylcellulose, hydroxypropyl methylcellulose, silica, silicadimethyl silylate, xanthan gum, and hydrogenatedbutylenes/ethylene/styrene copolymer.

Suitable moisturizers include, e.g., butylene glycol, cetyl alcohol,dimethicone, dimyristyl tartrate, glucose glycereth-26, glycerin,glyceryl stearate, hydrolyzed milk protein, lactic acid, lactose andother sugars, laureth-8, lecithin, octoxyglycerin, PEG-12, PEG 135,PEG-150, PEG-20, PEG-8, pentylene glycol, hexylene glycol, phytantriol,poly quaternium-39 PPG-20 methyl glucose ether, propylene glycol, sodiumhyaluronate, sodium lactate, sodium PCA, sorbitol, succinoglycan,synthetic beeswax, tri-C14-15 alkyl citrate, and starch.

In general, an enzyme composition as disclosed herein (e.g., acomposition including one or more Tgase variant enzyme(s), optionallyimmobilized on or encapsulated in a polymeric support, and sunscreen orcolor producing molecule(s)), can be incorporated into any suitablepersonal care product intended for use in protecting the skin from UVexposure or in modifying the appearance of skin, such as a cosmeticproduct (e.g., lipstick, foundation, blush, or eye makeup). Cosmeticproducts into which the disclosed compositions may be incorporatedinclude, but are not limited to, liquid or powder foundation, liquid orsolid eyeliner, blush, eye shadow, tinted powder, “pancake” type powderto be used dry or moistened, lip color, or makeup setting sprays, etc.The disclosed compositions may also be incorporated into a bronzer orartificial tanning product. Additionally, the disclosed compositions maybe incorporated into a sunscreen product, such as a chemical sunscreen,e.g., to bind a sunscreen chromophore (such as, but not limited to,oxybenzone, avobenzone, octisalate, octocrylene, homosalate, oroctinoxate, or a derivative thereof) to skin protein.

B. Household/Industrial Products

Non-limiting embodiments of household/industrial products which mayincorporate the disclosed Tgase variant enzymes or compositions thereofas disclosed herein as a preservative substance, either alone or incombination with one or more additional preservative substance, such asone or more petrochemically derived preservative substance, include, butare not limited to, householder cleaners, such as concentrated liquidcleaners and spray cleaners, cleaning wipes, dish washing liquid, dishwasher detergent, spray-mop liquid, furniture polish, indoor paint,outdoor paint, dusting spray, laundry detergent, fabric softener,rug/fabric cleaner, window and glass cleaner, toilet bowl cleaner,liquid/cream cleanser, etc. In a particular embodiment, the compositionsdescribed herein may be used in a food wash product, e.g., designed toclean fruits and vegetables prior to consumption. In some embodiments,one or more biobased preservative (i.e., Tgase variant enzyme orcomposition thereof as disclosed herein) is combined with one or moresynthetic preservative (e.g., petrochemically derived substance) and thepreservative (e.g., antimicrobial) effect achieved between the biobasedand synthetic preservatives is additive or synergistic.

C. Leather

In general, a Tgase enzyme, (e.g., Tgase variant enzyme as describedherein) and/or lysyl oxidase enzyme can be incorporated into any naturalcollagen containing product or used during leather processing to modifythe leather such that color is covalently bound to one or more proteinin leather, such as animal or non-animal derived collagen, keratin,silk, and/or elastin proteins.

D. Food Products

In general, a Tgase enzyme, (e.g., Tgase variant enzyme as describedherein) and/or lysyl oxidase enzyme can be incorporated into any foodprotein or used during food processing, to modify the color of foodprotein. Food products into which the Tgase and/or lysyl oxidaseenzyme(s) may be incorporated include, but are not limited to,animal-derived products containing collagen or gelatin (hydrolyzedcollagen). These include, but are not limited to, gelatin products, meatproducts or meat analogue products such as sausage casings, pork rinds,or any meat or marine product including the skin layer of the animaland/or collagen. In addition, the enzyme composition may be incorporatedinto non-animal derived collagen-containing products or anycollagen-containing product.

E. Other Products

Other products into which the disclosed Tgase variant enzymes orcompositions thereof as disclosed herein may be incorporated include,but are not limited to, food, pharmaceutical, cosmetic, healthcare,marine, paint, coating, adhesive, energy (e.g., fracking fluid),plastic, packaging, and agricultural products. In some embodiments, thedisclosed enzymes or enzyme-polymer compositions disclosed herein may beincorporated into HVAC systems, cooling ponds, water purificationsystems, or may be used in an industrial application, such as, but notlimited to, pulp and paper processing.

In some embodiments, a biocidal enzyme, i.e., Tgase variant enzyme asdisclosed herein, is combined with one or more additional preservativesubstance, such as one or more petrochemically derived preservativesubstance. In some embodiments, one or more biobased preservative (i.e.,Tgase variant enzyme or composition thereof as disclosed herein) iscombined with one or more synthetic preservative (e.g., petrochemicallyderived substance) and the preservative (e.g., antimicrobial) effectachieved between the biobased and synthetic preservatives is additive orsynergistic.

VII. Methods of Use

Methods are provided for use of the Tgase variants disclosed herein(including any of the variants disclosed in Table 3), optionally with anN-terminal methionine residue, including circular permutants thereof,and optionally with a pro-sequence as described herein) in variousapplications of use in which crosslinking of proteins or peptides isdesired or beneficial.

Tgase variants as described herein may be used in applications of usesuch as, but not limited to, preservative, antimicrobial, odor control,pharmaceutical, cosmetic, topical, industrial, energy, healthcare, ormarine applications.

The disclosed variants may be used as alternatives or in addition toconventional preservatives, such as, but not limited to, parabens,formaldehyde, and glutaraldehyde and conventional biocidal agents,including silver (used in wound care products), in various applicationsthat require preservatives for example, personal care, household,industrial, food, pharmaceutical, cosmetic, healthcare, marine, paint,coating, energy, plastic, packaging, and agricultural products, or inany of the products or systems disclosed herein. The disclosed variantsmay be used as anti-microbial (e.g., preservative) ingredients thatinhibit the growth of potentially harmful bacteria, fungi, and/or othermicrobes, and accordingly, are added to the product to be preserved inan effective amount to inhibit bacterial, fungal, and/or microbialgrowth in these products. Nonlimiting examples of such applications ofuse are described, for example, in PCT/US20/21211, which is incorporatedby reference herein in its entirety. The Tgase variants may be employedas antimicrobial agents with applications in healthcare products,personal care or cosmetic formulations, packaging (food, cosmetic, andpharmaceuticals), textile and leather production, paints and coatings,and marine applications including water treatment and purification.

The Tgase variants may be employed for permanently modifying proteins ofinterest, such as, but not limited to, keratin and collagen, withfunctional ingredients, dyes, or proteins. Tgase enzymes (such as any ofthe disclosed variants) and/or lysyl oxidase enzymes may be incorporatedinto products to facilitate covalent bonding of color, dye, or pigmentmolecules to proteins or peptides. The methods include contacting aprotein or material of interest with one or more Tgase enzyme (e.g.,Tgase variant as described herein) and/or lysyl oxidase enzyme and oneor more color-producing molecule, e.g., a dye or pigment molecule. TheTgase and/or lysyl oxidase enzyme(s) are present in an amount that issufficient (i.e., effective) to covalently bind the color molecule(s) tothe protein or material of interest. In some embodiments, the protein ofinterest is one or more protein present in skin, and the Tgase and/orlysyl oxidase enzyme(s) and/or color molecule(s) may be in the form of acosmetic or personal care product. The protein present in skin may becollagen, keratin, and/or elastin. In some embodiments, the protein ormaterial of interest is leather, a food product, or an agriculturalproduct, or a protein of interest therein, and the Tgase enzyme(s)(e.g., Tgase variant(s)) and/or lysyl oxidase) and color molecule(s) arein the form of a composition that is suitable for modifying or addingcolor to the leather, a food product, or an agricultural product, or aprotein of interest therein.

A. Preservative Methods

A Tgase variant as described herein (i.e., any of the variants disclosedin Table 3, optionally with an N-terminal methionine residue, includingcircular permutants thereof, and optionally with a pro-sequence asdescribed herein) may be used as an alternative to or in addition toconventional preservatives, such as, but not limited to, parabens,formaldehyde, and glutaraldehyde and conventional biocidal agents,including silver (used in wound care products), in various applicationsthat require preservatives for example, personal care, household,industrial, food, pharmaceutical, cosmetic, healthcare, marine, paint,coating, adhesive, energy, plastic, packaging, and agriculturalproducts. A Tgase variant may be used as an antimicrobial (e.g.,preservative) ingredient that inhibits the growth of potentially harmfulbacteria, fungi, and/or other microbes, and accordingly, is added to aproduct to be preserved in an effective amount to inhibit bacterial,fungal, and/or microbial growth in such a products. Nonlimiting examplesof such applications of use are described, for example, inPCT/US20/21211, which is incorporated by reference herein in itsentirety. In some embodiments, USP <51> passing criteria are achieved,i.e., for Category 2 Products: Bacteria: No less than 2.0 log reductionfrom the initial calculated count at 14 days, and no increase from the14 days' count at 28 days; for Yeast and Molds: No increase from theinitial calculated count at 14 and 28 days. In some embodiments, theantimicrobial behavior of the enzymes and enzyme-biopolymercoformulations are characterized by MIC (minimum inhibitoryconcentration) against gram-positive and gram-negative bacteria as wellas fungi, which results in reduction of microbial growth byapproximately 80-100%, or any of at least about 80%, 85%, 90%, 95%, 98%,or 99% of microbial growth.

When combined with a product as described herein, e.g., a personal care,household, industrial, food, pharmaceutical, cosmetic, healthcare,marine, paint, coating, adhesive, energy, plastic, packaging, oragricultural product, or in any of the products or systems disclosedherein, e.g., in a formulation or incorporated into a product or systemas a preservative, the composition may have effective broad spectrumpreservation activity over a broad pH range.

In some embodiments, the method includes adding a preservativecomposition as described herein (e.g., a Tgase variant or a compositionthereof as described herein) to a product or system, such as a personalcare, household, industrial, food, pharmaceutical, cosmetic, healthcare,marine, paint, coating, adhesive, energy, plastic, packaging, oragricultural product, or in any of the products or systems disclosedherein, e.g., in a formulation or incorporated into a product or system,wherein microbial growth is decreased and/or shelf life of the productis increased in comparison to an identical product that does not containthe preservative composition. In some embodiments, no other preservativeis included in the product composition, such as, but not limited toformaldehyde and/or glutaraldehyde.

In some embodiments, a method for increasing the shelf-life, integrity,or microbial free (e.g., bacterial and/or fungal free) status of aproduct composition, such as a personal care, household or industrialproduct is provided, wherein the method includes incorporating aneffective amount of a preservative composition as described herein intothe product (e.g., personal care, household or industrial product). Insome embodiments, the effective amount may be an amount, referred to asthe MIC (minimum inhibitory concentration), which results in reductionof microbial growth by approximately 80-100%, or any of at least about80%, 85%, 90%, 95%, 98%, or 99% reduction of microbial growth asdescribed herein.

In some embodiments of the methods or compositions described herein, theTgase variant enzyme may be included at a concentration of about 0.0001%w/v to about 1% w/v, 0.0001% w/v to about 0.01% w/v, about 0.0001% w/vto about 2.5% w/v, about 0.0001% w/v to about 5% w/v, about 0.0001% w/vto about 0.001% w/v, about 0.001% w/v to about 0.01% w/v, about 0.01%w/v to about 0.1% w/v, 0.01% w/v to about 5% w/v, or any of at leastabout 0.01% w/v, 0.05% w/v, 0.1% w/v, 0.5% w/v, 1% w/v, 1.5% w/v, 2%w/v, 2.5% w/v, 3% w/v, 3.5% w/v, 4% w/v, 4.5% w/v, or 5% w/v, or any ofabout 0.01% w/v to about 0.05% w/v, about 0.1% w/v to about 0.5% w/v,about 1% w/v to about 1.5% w/v, about 1.5% w/v to about 2% w/v, about 2%w/v to about 2.5% w/v, about 2.5% w/v to about 3% w/v, about 3% w/v toabout 3.5% w/v, about 3.5% w/v to about 4% w/v, about 4% w/v to about4.5% w/v, about 4.5% w/v to about 5% w/v, about 0.01% w/v to about 0.1%w/v, about 0.1% w/v to about 1% w/v, about 1% to about 5% w/v, about0.05% w/v to about 0.5% w/v, about 0.5% w/v to about 5% w/v, about 1%w/v to about 2.5% w/v, or about 2.5% w/v to about 5% w/v.

Non-limiting examples of personal care products to which thepreservative methods may be applied, utilizing the disclosed Tgasevariants and compositions thereof, include bar soap, liquid soap (e.g.,hand soap), hand sanitizer (including rinse off and leave-on alcoholbased and aqueous-based hand disinfectants), preoperative skindisinfectant, cleansing wipes, disinfecting wipes, body wash, acnetreatment products, antifungal diaper rash cream, antifungal skin cream,shampoo, conditioner, cosmetics (including but not limited to liquid orpowder foundation, liquid or solid eyeliner, mascara, cream eye shadow,tinted powder, “pancake” type powder to be used dry or moistened, makeup removal products etc.) deodorant, antimicrobial creams, body lotion,hand cream, topical cream, aftershave lotion, skin toner, mouth wash,toothpaste, sunscreen lotion, and baby products such as, but not limitedto, cleansing wipes, baby shampoo, baby soap, and diaper cream. Thepresent subject matter may also be applied to wound care items, such as,but not limited to, wound healing ointments, creams, and lotions, woundcoverings, burn wound cream, bandages, tape, and steri-strips, andmedical articles such as medical gowns, caps, face masks, andshoe-covers, surgical drops, etc. Additional products include but arenot limited to oral products such as mouth rinse, toothpaste, and dentalfloss coatings, veterinary and pet care products, preservativecompositions, and surface disinfectants including solutions, sprays orwipes.

Non-limiting examples of household/industrial products to which thepreservative methods may be applied, utilizing the disclosed Tgasevariants and compositions thereof, include householder cleaners such asconcentrated liquid cleaners and spray cleaners, cleaning wipes, dishwashing liquid, dish washer detergent, spray-mop liquid, furniturepolish, indoor paint, outdoor paint, dusting spray, laundry detergent,fabric softener, rug/fabric cleaner, window and glass cleaner, toiletbowl cleaner, liquid/cream cleanser, etc. In a particular embodiment,the preservative methods of the present subject matter may be used in afood wash product, designed to clean fruits and vegetables prior toconsumption, packaging, and food coatings.

B. Protein Modification Methods

In some embodiments, one or more Tgase enzyme, (e.g., Tgase variant asdisclosed herein) and/or lysyl oxidase enzyme may be included in aproduct to be used for long-lasting application of functionalingredients including UV-blocking sunscreens, and/or coloring agents,such as pigments or dyes. In some embodiments, any of the Tgase enzymesdisclosed in SEQ ID NOs:1-29, and/or a lysyl oxidase enzyme is includedin the product composition. For example, the Tgase (e.g., Tgase variant)and/or lysyl oxidase enzyme(s) may be used in a composition for deliveryof an active or functional ingredient to mammalian (e.g., human) skin,hair, or nails, such as, but not limited to, permanent (covalent) colormodification of the surface of hair fibers. In some embodiments, Tgase(e.g., Tgase variant) and/or lysyl oxidase enzyme(s) may be incorporatedin a product to be applied topically and which bonds to the skin of anindividual, such as a UV-blocking (sunscreen) product, or a cosmeticproduct. In some embodiments, the Tgase (e.g., Tgase variant) and/orlysyl oxidase enzyme(s) may be used to provide permanent application ofcolor to the skin of an animal such as in leather processing. In someembodiments, the Tgase (e.g., Tgase variant) and/or lysyl oxidaseenzyme(s) may be used to provide a permanent application of color infood processing.

Methods are provided herein for modifying or adding color to a proteinor material of interest. The methods include contacting a protein,peptide, or material of interest with one or more Tgase (e.g., Tgasevariant as described herein) and/or lysyl oxidase enzyme(s) and one ormore functional ingredient including a sunscreen and/or color-producingmolecule, e.g., a dye or pigment molecule. The Tgase (e.g., Tgasevariant) and/or lysyl oxidase enzyme(s) are present in an amount that issufficient (i.e., effective) to covalently bind the sunscreen and/orcolor molecule(s) to the protein, peptide, or material of interest.

In some embodiments, the protein of interest is one or more proteinpresent in skin, and the Tgase (e.g., Tgase variant) and/or lysyloxidase enzyme(s) and sunscreen(s) and/or color molecule(s) may be inthe form of a cosmetic or personal care product. The protein present inskin may be collagen, keratin, and/or elastin.

In some embodiments, the material of interest is one or more protein orpeptide derived from skin, and the Tgase (e.g., Tgase variant) and/orlysyl oxidase enzyme(s) and/or sunscreen(s) and/or color molecule(s) maybe in the form of a cosmetic or personal care product. The proteinpresent in the product formulation may be collagen, keratin, and/orelastin. The peptide present in the product formulation may behydrolyzed collagen, hydrolyzed keratin, and/or hydrolyzed elastin.

In some embodiments, the protein or material of interest is leather, afood product, or an agricultural product, or a protein of interesttherein, and the Tgase (e.g., Tgase variant) and/or lysyl oxidaseenzyme(s) and/or color molecule(s) are in the form of a composition thatis suitable for modifying or adding color to the leather, a foodproduct, or an agricultural product, or a protein of interest therein.

In some embodiments, a method is provided for delivering an active orfunctional ingredient (such as a sunscreen molecule or coloring agent)to proteins or peptides of mammalian (e.g., human) skin, hair, or nails.For example, the method may include application of a composition asdescribed herein to proteins or peptides of mammalian (e.g., human)skin, hair, or nails or topical application of the composition to skin,hair, or nails of a mammalian (e.g., human) individual.

In some embodiments, the method includes contacting proteins and/orpeptides of mammalian (e.g., human) skin, hair, or nails, with acomposition that includes: (a) an effective amount of at least oneactive or functional ingredient (such as, for example, a sunscreenmolecule or coloring agent); and (b) one or more Tgase (e.g., Tgasevariant) and/or lysyl oxidase enzyme(s) in an amount effective tocatalyze the crosslinking of the active or functional ingredient to aprotein or peptide of mammalian (e.g., human) skin, hair, or nails. Insome embodiments, the method includes topical application of thecomposition to the skin, hair, or nails of a mammalian (e.g., human)individual, and in certain embodiments the composition may contain: (c)a pharmaceutically or acceptable carrier in an amount effective todeliver the Tgase variant enzyme and the active or functional ingredientto the skin, hair, or nails of the individual.

For example, the active or functional ingredient may include at leastone alkylamino (—RNH₂), hydrazine, hydrazide, or hydroxylamine moiety,either directly on the active or functional ingredient, or indirectly ona linker attached (e.g., covalently bound) thereto, and the methodincludes catalysis by the transglutaminase enzyme of crosslinking (e.g.,formation of covalent bonds) between the amino groups of the active orfunctional ingredient and amino groups (e.g., amino groups on glutamineand/or lysine amino acid residue side chains) in proteins or peptides ofskin, hair, or nails.

The following examples are intended to illustrate, but not limit, theinvention.

EXAMPLES Example 1. Tgase Vector Construction and Mutagenesis Cell FreeProtein Synthesis (CFPS) Vectors

The genes coding for the pro-sequence and mature Tgase were codonoptimized for expression in E. coli based on the published amino acidsequence (Kanaji, et al. (1993) J. Biol. Chem. 268(16):11565-11572),synthesized, and cloned onto pUC19-derived expression vectors asdescribed in PCT/US20/49226.

E. coli Vector

The genes coding for the pro-sequence and mature Tgase were codonoptimized for expression in E. coli based on the published amino acidsequence (Kanaji, et al. (1993) J. Biol. Chem. 268(16):11565-11572) andsynthesized as described in PCT/US20/49226. The DNA was cloned onto thepET9a vector under control of the T7 promoter for expression in E. coli.

Creation of Tgase Variants

Mutations were introduced into the mature Tgase gene using site directedmutagenesis methods known in the art.

Example 2. Expression of Tgase Variants Using Cell-Free ProteinSynthesis

Pro-sequence and mature Tgase variants were expressed simultaneously ina commercially available cell-free protein synthesis kit following themanufacturer's instructions as described in PCT/US20/49226.

Example 3. Expression of Tgase Variants in E. coli

Single colonies of E. coli BL21(DE3) harboring the Tgase expressionplasmid were picked and used to inoculate 1 mL of LB in 96 deep wellplates. Starter cultures were grown overnight at 37° C., 400 rpm. Thefollowing morning, 100 μL of starter culture was used to inoculate 1 mLof media and incubated at 30° C., 400 rpm. After 6-8 hours, IPTG wasadded to a final concentration of 0.1 mM and the temperature was reducedto 20° C. Growth continued overnight and cultures were lysed and storedat −80° C.

Example 4. Measurement of Tgase Specific Activity

Tgase specific activity was measured in the examples herein using acolorimetric hydroxamate activity assay (Folk and Cole (1965) J BiolChemistry 240(7):2951-2960). Briefly, the hydroxamate assay usesN-carbobenzoxy-L-glutaminylglycine (Z-Gln-Gly or CBZ-Gln-Gly) as theamine acceptor substrate and hydroxylamine as an amine donor. In thepresence of transglutaminase, the hydroxylamine is incorporated to formZ-glutamylhydroxamate-glycine, which develops a colored complex withiron (III), detectable at 525 nm after incubation at 37° C. for 5-60minutes. The calibration was performed using L-glutamic acidγ-monohydroxamate (Millipore Sigma) as standard. One unit of Tgase isdefined as the amount of enzyme, determined using a commerciallyavailable ELISA kit (Zedira E021) following the manufacturer's protocol,that catalyzes formation of 1 μmol of the peptide derivative ofγ-glutamylhydroxylamine per minute.

Example 5. Protein Functionalization Assays

Protein functionalization by Tgase variants was determined by one of twomethods: (1) the fluorogenic labeling of casein usingmonodansylcadaverine; and (2) covalently locking a fluorescent substrateonto a collagen plate. All results were normalized to Tgaseconcentration using ELISA.

Fluorogenic Casein-Labeling Assay

Initial rates of the active mutants for protein substrates weredetermined by measuring the increase in fluorescence over timeassociated with Tgase-catalyzed labeling of casein with dansylcadaverine(e.g., a commercially available kit, Fluorogenic Activity Assay Kit,Zedira T036).

Fluorescent Collagen-Labeling Assay

Amine donor dye. TAMRA-cadaverine (1.7 g/L) was covalently bound to acollagen plate (Corning BioCoat Collagen I Multiwell Plates) in thepresence of increasing amounts of Tgase, SEQ ID NO:28. Tgase variant(0-0.33% w/v) in phosphate buffered saline (PBS) at pH 7.4, total volumeof 60 μL. A negative control containing bovine serum albumin (BSA, 0.33%w/v) in the absence of Tgase was run in parallel. The plate wasincubated for 16 h at 37° C.

A scheme of the reaction is illustrated in FIG. 7 . The results areshown in FIG. 9 . The wells are shown pre-color removal (top row) andpost-wash with PBS (bottom row).

Glutamine donor dye. Cbz-Gln-Gly-TAMRA (2.5 g/L) was bonded to acollagen plate (Corning BioCoat Collagen I Multiwell Plates) in thepresence of Tgase with the amino acid sequence depicted in SEQ ID NO:28(0.05-0.3% w/v) in PBS at pH 7.4. A negative control containing BSA(0.33% w/v) and a negative control in the absence of BSA or Tgase wererun in parallel. The plate was incubated for 16 h at 37° C. Followingincubation, the plates were washed with PBS to remove any residual,unbound dye.

A scheme of the reaction is illustrated in FIG. 8 . The results areshown in FIG. 10 . The wells are shown pre-color removal and post-washwith PBS.

Example 6. Antimicrobial Activity Assays Minimum InhibitoryConcentration (MIC) Assay

Yeast or bacterial starter cultures were grown at 30° C.-37° C.overnight. The following day, the cell density of the saturated cultureswas calculated using OD600 and cultures were diluted to 10⁵-10⁸ cellsper mL. Cultures (100 μL) were made from the dilute starters in 96 wellplates. Mutant or wild-type Tgase was added to each culture at 0.0001-1weight percent. The cultures were grown overnight at 30° C.-37° C. andgrowth curves were measured by a BioTek Synergy Plate Reader. Thefollowing day, a cell viability assay such as BacTiter Glo (Promegafollowing manufacturer's protocols) was used to assess cell survivalrate following challenge with Tgase. A decrease in luminescenceindicates a decrease in cell viability. Results are shown in Table 3.

Cell Growth (OD Measurement)

E. coli was cultured in the presence of wild-type Tgase or Tgasevariants at concentrations ranging from 1 mg/L-250 mg/L. Lower opticaldensity (OD) measurements correlated to improvements in antibacterialefficacy relative to wild-type Tgase.

TABLE 3 Activity Assay Results for Tgase Variants Protein Variant SEQ.Mutations (Relative to Specific Functionalization Antimicrobial Name ID.NO. SEQ. ID. NO. 1) Activity Activity Activity M2 2 S199G S299V ++ ++N/A M3 3 H289V S299A ++ ++ N/A M4 4 N282M H289T S299V ++ N/A N/A M5 5N282E H289V S299K ++ ++ + M6 6 S284D H289L S299K ++ N/A N/A M7 7 N282EH289I S299K ++ +++ +++ M8 8 N282K G283A S299V + + + M9 9 N282Q S284PH289E S299V ++ N/A N/A M10 10 N282K G283A S284P S299V ++ − +++ M11 11N282R G283A S284E H289Q S299V +++ ++ + M12 12 S199A S299A ++ − +++ M1313 S199A S299E ++ ++ N/A M14 14 S199A S299K ++ ++ + M15 15 S199A S299V+++ +++ +++ M16 16 S199G S299A ++ ++ N/A M17 17 S199G S299K ++ N/A N/AM18 18 S2P S199A S299A + N/A N/A M19 19 S2P S199A S299E ++ ++ N/A M20 20S2P S199A S299K ++ ++ + M21 21 S2P S199A S299V ++ ++ + M22 22 S2P S199GS299A ++ + + M23 23 S2P S199G S299E + N/A N/A M24 24 S2P S199G S299K ++++ + M25 25 S2P S199G S299V +++ +++ ++ M26 26 N282D G283A S284A S299V ++++ N/A M27 27 S199G S299E +++ ++ + M28 28 S2P + ++ + Variants ofStreptomyces mobaraensis Tgase and improvements to activity relative towild-type S. mobaraensis Tgase. Numbering of amino acid positions is inreference to the mature S. mobaraensis Tgase amino acid sequencedepicted in SEQ ID NO: 1. A “−” indicates a reduction in activity. A “+”indicates an improvement between 1.2- and 2-fold. A “++” indicates animprovement between 2- and 5-fold. A “+++” indicates an improvementgreater than 5-fold.

Example 7. Formation of Sunscreen-Linker

Oxybenzone (0.25 mol), toluene (100 mL), and glacial acetic acid (1drop) were charged in a 250 mL three neck flask fitted with a mechanicalagitator, thermocouple, and dean stark trap. Molten 1,6-hexanediamine(0.25 mol) was added to the flask and the reaction was refluxedovernight to remove water. The reaction was monitored by HPLC. Theresulting oxybenzone-imine was isolated by rotary evaporation underreduced pressure and further dried under vacuum to deliver the imineproduct, 2-(((6-aminohexyl)imino)(phenyl)methyl)-5-methoxyphenol, as ayellow solid in nearly quantitative yield (m/z 327.3). The scheme isshown in FIG. 5 .

Example 8. Covalent Addition of Sunscreen-Linker to Protein

The imine product in Example 7 (1.6 g/L) and Cbz-Gln-Gly dipeptide (1.65g/L) were dissolved in 0.1M Tris-HCl pH 8.0. To aid in dissolution ofthe imine, 10% 1:1 dichloromethane in dimethyl sulfoxide was employed.Tgase (0.01 wt %) was added to the suspension and the reaction wasincubated at 37° C. overnight with constant agitation. Tgase catalyzed,covalent addition of the dipeptide to the imine was confirmed by LCMS(m/z 647.25). In the absence of Tgase, no covalent addition wasobserved. The scheme is shown in FIG. 5 and results are shown in FIG. 6. Tgase variant, SEQ ID NO:28 demonstrates 11-fold improvement inactivity relative to wild-type Tgase. No product was observed in theabsence of Tgase (negative control).

Amino Acid SequencesSEQ ID NO: 1 Wild-Type Tgase from Streptomyces mobaraensisDSDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYSKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWSEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP SEQ ID NO: 2 S199G S299VDSDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYGKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWVEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP SEQ ID NO: 3 H289V S299ADSDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYSKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMVVYESKFRNWAEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP SEQ ID NO: 4 N282M H289T S299VDSDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYSKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPMGSLGAMTVYESKFRNWVEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP SEQ ID NO: 5 N282E H289V S299KDSDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYSKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPEGSLGAMVVYESKFRNWKEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP SEQ ID NO: 6 S284D H289L S299KDSDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYSKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGDLGAMLVYESKFRNWKEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP SEQ ID NO: 7 N282E H289I S299KDSDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYSKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPEGSLGAMIVYESKFRNWKEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP SEQ ID NO: 8 N282K G283A S299VDSDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYSKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPKASLGAMHVYESKFRNWVEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP SEQ ID NO: 9 N282Q S284P H289E S299VDSDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYSKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPQGPLGAMEVYESKFRNWVEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWPSEQ ID NO: 10 N282K G283A S284P S299VDSDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYSKHFWSGQDRSSSADKRKYGDPDAERPAPGTGLVDMSRDRNIPRSPTSPGEGEVNFDYGWFGAQTEADADKTVWTHGNHYHAPKAPLGAMHVYESKERNWVEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWPSEQ ID NO: 11 N282R G283A S284E H289Q S299VDSDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYSKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPRAELGAMQVYESKFRNWVEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP SEQ ID NO: 12 S199A S299ADSDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYAKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWAEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP SEQ ID NO: 13 S199A S299EDSDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYAKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWEEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP SEQ ID NO: 14 S199A S299KDSDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYAKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWKEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP SEQ ID NO: 15 S199A S299VDSDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYAKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWVEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP SEQ ID NO: 16 S199G S299ADSDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYGKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWAEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP SEQ ID NO: 17 S199G S299KDSDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYGKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWKEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP SEQ ID NO: 18 S2P S199A S299ADPDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYAKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWAEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP SEQ ID NO: 19 S2P S199A S299EDPDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYAKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWEEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP SEQ ID NO: 20: S2P S199A S299KDPDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYAKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWKEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP SEQ ID NO: 21 S2P S199A S299VDPDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYAKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWVEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP SEQ ID NO: 22 S2P S199G S299ADPDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYGKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWAEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP SEQ ID NO: 23 S2P S199G S299EDPDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYGKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWEEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP SEQ ID NO: 24 S2P S199G S299KDPDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYGKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWKEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP SEQ ID NO: 25 S2P S199G S299VDPDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYGKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWVEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWPSEQ ID NO: 26 N282D G283A S284A S299VDSDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYSKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPDAALGAMHVYESKFRNWVEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP SEQ ID NO: 27 $199G S2992DSDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYGKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWEEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP SEQ ID NO: 28 S2PDPDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYSKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWSEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWPSEQ ID NO: 29 Wild-Type Tgase with polyhistidine tag.DSDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYSKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWSEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWPLEHHHHHH

We claim:
 1. A variant of the transglutaminase enzyme amino acidsequence depicted in SEQ ID NO:1, comprising substitutions of amino acidresidue A or G at position 199 and amino acid residue A, E, K, or V atposition
 299. 2. The transglutaminase variant of claim 1, furthercomprising an N-terminal methionine residue, a C-terminal histidinepolyhistidine sequence, and/or a pro-sequence.
 3. The transglutaminasevariant of claim 1, further comprising substitution of amino acidresidue C, D, E, F, H, I, K, L, M, N, P, Q, R, T, V, W, Y at position 2.4. The transglutaminase variant of claim 3, further comprising anN-terminal methionine residue, a C-terminal histidine polyhistidinesequence, and/or a pro-sequence.
 5. A variant of the transglutaminaseenzyme amino acid sequence depicted in SEQ ID NO:1, comprisingsubstitutions of amino acid residue D, E, K, M, R, or Q at position 282,amino acid residue E, I, L, T, Q, or V at position 289, and amino acidresidue A, E, K, or V at position
 299. 6. The transglutaminase variantof claim 5, further comprising an N-terminal methionine residue, aC-terminal histidine polyhistidine sequence, and/or a pro-sequence.
 7. Avariant of the transglutaminase enzyme amino acid sequence depicted inSEQ ID NO:1, comprising the amino acid sequence depicted in any of SEQID NOs: 2-27.
 8. The transglutaminase variant of claim 7, furthercomprising an N-terminal methionine residue, a C-terminal histidinepolyhistidine sequence, and/or a pro-sequence.
 9. A transglutaminaseenzyme, comprising a sequence having at least about 90% sequenceidentity to any of the amino acid sequences depicted in SEQ ID NOs:2-27or a circular permutant of any of the amino acid sequences depicted inSEQ ID NOs: 2-27, wherein the variant comprises at least about 2-foldgreater transglutaminase enzymatic activity than the transglutaminaseenzyme comprising the amino acid sequence depicted in SEQ ID NO:1. 10.The transglutaminase variant of claim 9, further comprising anN-terminal methionine residue, a C-terminal histidine polyhistidinesequence, and/or a pro-sequence.
 11. A method for increasing the shelflife of a product, comprising incorporating the transglutaminase variantof any of claims 1 to 10 into the product in an amount effective toprevent or decrease growth of one or more microbe in comparison to anidentical product that does not comprise the variant.
 12. A productcomprising the transglutaminase variant of any of claims 1 to 10 in aneffective amount to increase the shelf life of the product, incomparison to an identical product that does not comprise the variant.13. The product of claim 12, wherein the product is a personal care,household, industrial, food, pharmaceutical, cosmetic, healthcare, woundcare, marine, paint, coating, energy, plastic, packaging, textile,leather, or agricultural product.
 14. The product of 13, wherein theproduct is a personal care product selected from bar soap, liquid soap,hand sanitizer, preoperative skin disinfectant, cleansing wipes,disinfecting wipes, body wash, acne treatment products, antifungaldiaper rash cream, antifungal skin cream, shampoo, conditioner,cosmetic, deodorant, antimicrobial cream, body lotion, hand cream,topical cream, aftershave lotion, skin toner, mouth wash, toothpaste,and sunscreen lotion.
 15. The product of claim 13, wherein the productis a wound care product selected from wound healing ointment, cream, andlotion, wound covering, burn wound cream, bandages, tape, andsteri-strips.
 16. An enzyme composition comprising: (i) thetransglutaminase variant of any of claims 1 to 10, a transglutaminaseenzyme comprising the amino acid sequence depicted in SEQ ID NO:1, 28,or 29, and/or a lysyl oxidase enzyme; and (ii) a substrate for thetransglutaminase or lysyl oxidase enzyme, comprising a sunscreenmolecule, a pigment or dye molecule, or a functional ingredientmolecule.
 17. The enzyme composition of claim 16, wherein the sunscreenmolecule, pigment or dye molecule, or functional ingredient molecule isconjugated to a molecule that comprises a free amino group.
 18. Theenzyme composition of claim 17, wherein the molecule that comprises afree amino group is derived from an aliphatic amine of formulaR(CH₂)_(n)(NH₂), wherein n is an integer between 1 and 30 and R is afunctional ingredient.
 19. The enzyme composition of claim 18, wherein nis an integer between 5 and
 10. 20. The enzyme composition of claim 17,wherein the molecule that comprises a free amino group is derived fromlysine, cadaverine, putrescine, hydrazine, adipic acid dihydrazide,sebacic dihydrazide, and hexamethylenediamine.
 21. The enzymecomposition of claim 16, wherein the sunscreen molecule, pigment or dyemolecule, or functional ingredient molecule is conjugated to an aminoacid, peptide, or protein with a free glutamine side chain.
 22. Acosmetic composition comprising the enzyme composition of claim
 16. 23.A method for bonding color to a material or protein of interest,comprising contacting the material or protein of interest with thetransglutaminase variant of any of claims 1 to 10, a transglutaminaseenzyme comprising the amino acid sequence depicted in SEQ ID NO:1, 28,or 29, and/or a lysyl oxidase enzyme and a pigment or dye molecule,wherein the transglutaminase or lysyl oxidase enzyme is present in anamount effective to covalently bind the pigment or dye molecule to thematerial or protein of interest.
 24. The method of claim 23, wherein theprotein of interest is a protein that is present in skin.
 25. The methodof claim 24, wherein the protein that is present in skin comprisescollagen, keratin, and/or elastin.
 26. A product comprising thetransglutaminase variant of any of claims 1 to 10, a transglutaminaseenzyme comprising the amino acid sequence depicted in SEQ ID NO:1, 28,or 29, and/or a lysyl oxidase enzyme in an effective amount to add afunctional ingredient or color molecule onto a protein or a protein-,peptide-, or amino acid-containing material of interest when contactedwith the product.
 27. The product of claim 26, wherein the product is apersonal care, cosmetic, textile, leather, food, or agriculturalproduct.
 28. A method of modifying the color of a protein or material ofinterest, comprising contacting the protein or material of interest withthe product according to claim
 26. 29. A composition comprising thetransglutaminase variant of any of claims 1 to 10 in combination withone or more antimicrobial enzyme, peptide, or protein, wherein thecomposition comprises preservative, biocidal, antimicrobial, orvirucidal activity.
 30. The composition of claim 29, wherein theantimicrobial enzyme, peptide, or protein is selected from lysozyme,chitinase, lipase, lysin, lysostaphin, glucanase, DNase, RNase,lactoferrin, glucose oxidase, peroxidase, lactoperoxidase, lactonase,acylase, dispersin B, a-amylase, cellulase, nisin, bacteriocin,siderophore, polymyxin, and defensin.
 31. A bacteriophage, comprising anucleic acid sequence that encodes the transglutaminase variant of anyof claims 1 to
 10. 32. A composition comprising the bacteriophage ofclaim 31, wherein the composition comprises antimicrobial activity. 33.The composition of claim 32, wherein the composition further comprises apharmaceutically acceptable excipient.